U.S. patent application number 14/952786 was filed with the patent office on 2016-07-28 for heterodimeric antibodies that bind cd3 and cd38.
The applicant listed for this patent is Xencor, Inc.. Invention is credited to Matthew J. Bernett, Seung Chu, John Desjarlais, Gregory Moore, Umesh Muchhal, Rumana Rashid.
Application Number | 20160215063 14/952786 |
Document ID | / |
Family ID | 55022681 |
Filed Date | 2016-07-28 |
United States Patent
Application |
20160215063 |
Kind Code |
A1 |
Bernett; Matthew J. ; et
al. |
July 28, 2016 |
HETERODIMERIC ANTIBODIES THAT BIND CD3 AND CD38
Abstract
The present invention is directed to heterodimeric antibodies
that bind CD3 and CD38.
Inventors: |
Bernett; Matthew J.;
(Monrovia, CA) ; Moore; Gregory; (Azusa, CA)
; Desjarlais; John; (Pasadena, CA) ; Chu;
Seung; (Cypress, CA) ; Rashid; Rumana; (Temple
City, CA) ; Muchhal; Umesh; (Monrovia, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Xencor, Inc. |
Monrovia |
CA |
US |
|
|
Family ID: |
55022681 |
Appl. No.: |
14/952786 |
Filed: |
November 25, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62085106 |
Nov 26, 2014 |
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62250971 |
Nov 4, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2809 20130101;
C07K 16/2896 20130101; C07K 2317/526 20130101; C07K 2317/524
20130101; C07K 2317/622 20130101; C07K 2317/73 20130101; C07K
2317/31 20130101; C07K 16/3061 20130101; C07K 2317/24 20130101;
A61P 35/02 20180101; C07K 16/18 20130101; C07K 2317/565 20130101;
A61K 2039/505 20130101; C07K 2317/64 20130101; C07K 2317/33
20130101; C07K 2317/522 20130101; C07K 2317/55 20130101; C07K
2317/567 20130101; C07K 2317/92 20130101; C07K 16/40 20130101; C07K
2317/56 20130101; A61P 35/00 20180101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; C07K 16/28 20060101 C07K016/28 |
Claims
1.-58. (canceled)
59. A heterodimeric anti-CD3 and anti-CD38 antibody comprising: A.
a) a first monomer comprising SEQ ID NO:91; b) a second monomer
comprising SEQ ID NO:92; and c) a light chain comprising SEQ ID
NO:93; or B. a) a first monomer comprising SEQ ID NO:88; b) a
second monomer comprising SEQ ID NO:89; and c) a light chain
comprising SEQ ID NO:90; or C. a) a first monomer comprising: i) a
first Fc domain; ii) an anti-CD3 scFv comprising a scFv variable
light domain, an scFv linker and a scFv variable heavy domain;
wherein said scFv is covalently attached to the N-terminus of said
Fc domain using a domain linker; b) a second monomer comprising a
heavy chain comprising: i) a heavy variable domain; and ii) a heavy
constant domain comprising a second Fc domain; and c) a light chain
comprising a variable light domain and a constant light domain;
wherein said scFv variable light domain comprises: the vlCDRs of
CD3 L1.47 comprising a vlCDR1 having SEQ ID NO:15, a vlCDR2 having
SEQ ID NO:16 and a vlCDR3 having SEQ ID NO:17, said scFv variable
heavy domain comprises the vlCDRs of CD3 H1.32 comprising a vhCDR1
having SEQ ID NO:11, a vhCDR2 having SEQ ID NO:12 and a vhCDR3
having SEQ ID NO:13, and wherein said heavy variable domain and
said variable light domain bind CD38; or D. a) a first monomer
comprising: i) a first Fc domain; ii) an anti-CD3 scFv comprising a
scFv variable light domain, an scFv linker and a scFv variable
heavy domain; wherein said scFv is covalently attached to the
N-terminus of said Fc domain using a domain linker; b) a second
monomer comprising a heavy chain comprising: i) a heavy variable
domain; and ii) a heavy constant domain comprising a second Fc
domain; and c) a light chain comprising a variable light domain and
a constant light domain; wherein said scFv variable light domain
comprises: the vlCDRs of CD3 L1.47, comprising a vlCDR1 having SEQ
ID NO:24, a vlCDR2 having SEQ ID NO:25 and a vlCDR3 having SEQ ID
NO:26, said scFv variable heavy domain comprises the vhCDRs of CD3
H1.89 comprising a vhCDR1 having SEQ ID NO:20, a vhCDR2 having SEQ
ID NO:21 and a vhCDR3 having SEQ ID NO:22, and wherein said heavy
variable domain and said variable light domain bind CD38; or E. a)
a first monomer comprising: i) a first Fc domain; ii) an anti-CD3
scFv comprising a scFv variable light domain, an scFv linker and a
scFv variable heavy domain; wherein said scFv is covalently
attached to the N-terminus of said Fc domain using a domain linker;
b) a second monomer comprising a heavy chain comprising: i) a heavy
variable domain; and ii) a heavy constant domain comprising a
second Fc domain; and c) a light chain comprising a variable light
domain and a constant light domain; wherein said scFv variable
light domain comprises: the vlCDRs of CD3 L1.47, comprising a
vlCDR1 having SEQ ID NO:24, a vlCDR2 having SEQ ID NO:25 and a
vlCDR3 having SEQ ID NO:26, said scFv variable heavy domain
comprises the vhCDRs of CD3 H1.89 comprising a vhCDR1 having SEQ ID
NO:20, a vhCDR2 having SEQ ID NO:21 and a vhCDR3 having SEQ ID
NO:22, and wherein said heavy variable domain and said variable
light domain bind CD38; or F. a) a first monomer comprising: i) a
first Fc domain; ii) an anti-CD3 scFv comprising a scFv variable
light domain, an scFv linker and a scFv variable heavy domain;
wherein said scFv is covalently attached to the N-terminus of said
Fc domain using a domain linker; b) a second monomer comprising a
heavy chain comprising: i) a heavy variable domain; and ii) a heavy
constant domain comprising a second Fc domain; and c) a light chain
comprising a variable light domain and a constant light domain;
wherein said scFv variable light domain comprises: the vlCDRs of
CD3 L1.47, comprising a vlCDR1 having SEQ ID NO:42, a vlCDR2 having
SEQ ID NO:43 and a vlCDR3 having SEQ ID NO:44, said scFv variable
heavy domain comprises the vhCDRs of H1.33 comprising a vhCDR1
having SEQ ID NO:38, a vhCDR2 having SEQ ID NO:39 and a vhCDR3
having SEQ ID NO:40, and wherein said heavy variable domain and
said variable light domain bind CD38; or G. a) a first monomer
comprising: i) a first Fc domain; ii) an anti-CD3 scFv comprising a
scFv variable light domain, an scFv linker and a scFv variable
heavy domain; wherein said scFv is covalently attached to the
N-terminus of said Fc domain using a domain linker; b) a second
monomer comprising a heavy chain comprising: i) a heavy variable
domain; and ii) a heavy chain constant domain comprising a second
Fc domain; and c) a light chain comprising a variable light domain
and a variable light constant domain; wherein said variable light
domain comprises a vlCDR1 having the sequence RASQNVDTWVA (SEQ ID
NO:60), a vlCDR2 having the sequence SASYRYS (SEQ ID NO:61) and a
vlCDR3 having the sequence QQYDSYPLT (SEQ ID NO:62), said variable
heavy domain comprising a vhCDR1 having the sequence YSWMN (SEQ ID
NO:56, a vhCDR2 having the sequence EINPQSSTINYATSVKG (SEQ ID
NO:57) and a vhCDR3 having the sequence YGNWFPY (SEQ ID NO:58); or
H. a) a first monomer comprising: i) a first Fc domain; ii) an
anti-CD3 scFv comprising a scFv variable light domain, an scFv
linker and a scFv variable heavy domain; wherein said scFv is
covalently attached to the N-terminus of said Fc domain using a
domain linker; b) a second monomer comprising a heavy chain
comprising: i) a heavy variable domain; and ii) a heavy chain
constant domain comprising a second Fc domain; and c) a light chain
comprising a variable light domain and a variable light constant
domain; wherein said variable light domain comprises: a vlCDR1
having the sequence RASQNVDTWVA (SEQ ID NO:69), a vlCDR2 having the
sequence SASYRYS (SEQ ID NO:70) and a vlCDR3 having the sequence
QQYDSYPLT (SEQ ID NO:71), said variable heavy domain comprises a
vhCDR1 having the sequence RSWMN (SEQ ID NO:65), a vhCDR2 having
the sequence EINPDSSTINYATSVKG (SEQ ID NO:66) and a vhCDR3 having
the sequence YGNWFPY (SEQ ID NO:67); or I. a) a first monomer
comprising: i) a first Fc domain; ii) an anti-CD3 scFv comprising a
scFv variable light domain, an scFv linker and a scFv variable
heavy domain; wherein said scFv is covalently attached to the
N-terminus of said Fc domain using a domain linker; b) a second
monomer comprising a heavy chain comprising: i) a heavy variable
domain; and ii) a heavy chain constant domain comprising a second
Fc domain; and c) a light chain comprising a variable light domain
and a variable light constant domain; wherein said variable light
domain comprises: a vlCDR1 having the sequence RASQNVDTNVA (SEQ ID
NO:78), a vlCDR2 having the sequence SASYRYS (SEQ ID NO:79) and a
vlCDR3 having the sequence QQYDSYPLT (SEQ ID NO:80), said variable
heavy domain comprises a vhCDR1 having the sequence RSWMN (SEQ ID
NO:74), a vhCDR2 having the sequence EINPDSSTINYATSVKG (SEQ ID
NO:75) and a vhCDR3 having the sequence YGNWFPY (SEQ ID NO:76).
60. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part C, D, E, or F wherein said variable light domain comprises: a
vlCDR1 having the sequence RASQNVDTWVA (SEQ ID NO:69), a vlCDR2
having the sequence SASYRYS (SEQ ID NO:70) and a vlCDR3 having the
sequence QQYDSYPLT (SEQ ID NO:71), said variable heavy domain
comprises a vhCDR1 having the sequence RSWMN (SEQ ID NO:65), a
vhCDR2 having the sequence EINPDSSTINYATSVKG (SEQ ID NO:66) and a
vhCDR3 having the sequence YGNWFPY (SEQ ID NO:67).
61. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part C, D, E, or F wherein said variable light domain has the
sequence
DIVMTQSPSSLSASVGDRVTITCRASQNVDTWVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTL-
TISSLQPEDFATYFCQQYDSYPLTFGGGTKLEIK (SEQ ID NO:68) and said variable
heavy domain has the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRSWMNWVRQAPGKGLEWVSEINPDSSTINYATSVKGRFTISRD-
NSKNTLYLQMNSLRAEDTAVYYCARYGNWFPYWGQGTLVTVSS (SEQ ID NO:64).
62. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part C, D, E, or F wherein said variable light domain comprises:
the vlCDRs of OKT10 L1 comprising a vlCDR1 having the sequence
RASQNVDTNVA (SEQ ID NO:78), a vlCDR2 having the sequence SASYRYS
(SEQ ID NO:79) and a vlCDR3 having the sequence QQYDSYPLT (SEQ ID
NO:80), said variable heavy domain comprises the OKT10 H1 comprises
a vhCDR1 having the sequence RSWMN (SEQ ID NO:74), a vhCDR2 having
the sequence EINPDSSTINYATSVKG (SEQ ID NO:75) and a vhCDR3 having
the sequence YGNWFPY (SEQ ID NO:76).
63. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part C, D, E, or F wherein said variable light domain has the
sequence SEQ ID NO:77 and said variable heavy domain has the
sequence SEQ ID NO:73.
64. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part C, D, E, or F wherein said variable light domain comprises:
the vlCDRs of OKT10L1.24 comprising a vlCDR1 having the sequence
RASQNVDTWVA (SEQ ID NO:60), a vlCDR2 having the sequence SASYRYS
(SEQ ID NO:61) and a vlCDR3 having the sequence QQYDSYPLT (SEQ ID
NO:62), said variable heavy domain the vlCDRs of OKT10H1.77
comprising a vhCDR1 having the sequence YSWMN (SEQ ID NO:56), a
vhCDR2 having the sequence EINPQSSTINYATSVKG (SEQ ID NO:57) and a
vhCDR3 having the sequence YGNWFPY (SEQ ID NO:58).
65. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part C, D, E, or F wherein said variable light domain has the
sequence SEQ ID NO:59 and said variable heavy domain has the
sequence SEQ ID NO:55.
66. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part H wherein said variable light domain has the sequence
DIVMTQSPSSLSASVGDRVTITCRASQNVDTWVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTL-
TISSLQPEDFATYFCQQYDSYPLTFGGGTKLEIK (SEQ ID NO:68) and said variable
heavy domain has the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRSWMNWVRQAPGKGLEWVSEINPDSSTINYATSVKGRFTISRD-
NSKNTLYLQMNSLRAEDTAVYYCARYGNWFPYWGQGTLVTVSS (SEQ ID NO:64).
67. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part I wherein said variable light domain has the sequence SEQ ID
NO:77 and said variable heavy domain has the sequence SEQ ID
NO:73.
68. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part G, H, or I wherein said scFv has sequence SEQ ID NO:18.
69. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part G, H, or I wherein said scFv has the sequence SEQ ID
NO:27.
70. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part G, H, or I wherein said scFv has the sequence SEQ ID
NO:36.
71. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part G, H, or I wherein said scFv has the sequence SEQ ID
NO:45.
72. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59
part G, H, or I wherein said scFv has the sequence SEQ ID
NO:54.
73. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59,
wherein said first Fc domain and said second Fc domain comprise a
set of variants selected from the group consisting of
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L and
K370S:S364K/E357Q.
74. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59,
wherein said scFv linker is a charged linker.
75. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59,
wherein said heavy chain constant domain comprises the amino acid
substitutions N208D/Q295E/N384D/Q418E/N421D.
76. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 59,
wherein said first and second Fc domains comprise the amino acid
substitutions E233P/L234V/L235A/G236del/S267K.
77. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 73,
wherein said scFv has a polypeptide sequence selected from the
group consisting of SEQ ID NO:9 (scFv 13551), SEQ ID NO:45 (scFv
15426), SEQ ID NO:9 (scFv 13243) and SEQ ID NO:54 (scFv 14702).
78. The heterodimeric anti-CD3 and anti-CD38 antibody of claim 73,
wherein said first variable heavy domain and said variable light
domain are a pair of domains selected from the pairs of domains
consisting of SEQ ID NO:55 and SEQ ID NO:59 (vh and vl 13551); SEQ
ID NO:55 and SEQ ID NO:59 (vh and vl 15426); SEQ ID NO:73 and SEQ
ID NO:77 (vh and vl 13243) and SEQ ID NO:55 and SEQ ID NO:59 (vh
and vl 14702).
79. A nucleic acid composition encoding the heterodimeric antibody
of claim 59, said composition comprising: a) a first nucleic acid
encoding said first monomer; b) a second nucleic acid encoding said
second monomer; and c) a third nucleic acid encoding said light
chain.
80. The nucleic acid composition of claim 79 comprising: a) a first
nucleic acid encoding SEQ ID NO:91; b) a second nucleic acid
encoding SEQ ID NO:92; and c) a third nucleic acid encoding SEQ ID
NO:93.
81. The nucleic acid composition of claim 79 comprising: a) a first
nucleic acid encoding SEQ ID NO:88; b) a second nucleic acid
encoding SEQ ID NO:89; and c) a third nucleic acid encoding SEQ ID
NO:90.
82. An expression vector composition comprising: a) an expression
vector comprising a nucleic acid encoding a first monomer of claim
59; b) an expression vector comprising a nucleic acid encoding said
second monomer of claim 59; and c) an expression vector comprising
a nucleic acid encoding said light chain of claim 59.
83. A host cell comprising the nucleic acid composition of claim
80.
84. A host cell comprising the expression vector composition of
claim 82.
85. A method of making a heterodimeric anti-CD3 and anti-CD38
antibody of claim 59 comprising culturing the host cell of claim 83
under conditions wherein said antibody is expressed, and recovering
said antibody.
86. A method of making a heterodimeric anti-CD3 and anti-CD38
antibody of claim 59 comprising culturing the host cell of claim 24
under conditions wherein said antibody is expressed, and recovering
said antibody.
87. A method of treating cancer comprising administering
heterodimeric anti-CD3 and anti-CD38 antibody of claim 59 to a
patient in need thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 62/085,106,
filed Nov. 26, 2014 and U.S. Provisional Patent Application No.
62/250,971, filed Nov. 4, 2015, all of which are expressly
incorporated herein by reference in their entirety, with particular
reference to the figures, legends and claims therein.
BACKGROUND OF THE INVENTION
[0002] Antibody-based therapeutics have been used successfully to
treat a variety of diseases, including cancer and
autoimmune/inflammatory disorders. Yet improvements to this class
of drugs are still needed, particularly with respect to enhancing
their clinical efficacy. One avenue being explored is the
engineering of additional and novel antigen binding sites into
antibody-based drugs such that a single immunoglobulin molecule
co-engages two different antigens. Such non-native or alternate
antibody formats that engage two different antigens are often
referred to as bispecifics. Because the considerable diversity of
the antibody variable region (Fv) makes it possible to produce an
Fv that recognizes virtually any molecule, the typical approach to
bispecific generation is the introduction of new variable regions
into the antibody.
[0003] A number of alternate antibody formats have been explored
for bispecific targeting (Chames & Baty, 2009, mAbs 1[6]:1-9;
Holliger & Hudson, 2005, Nature Biotechnology 23[9]:1126-1136;
Kontermann, mAbs 4(2):182 (2012), all of which are expressly
incorporated herein by reference). Initially, bispecific antibodies
were made by fusing two cell lines that each produced a single
monoclonal antibody (Milstein et al., 1983, Nature 305:537-540).
Although the resulting hybrid hybridoma or quadroma did produce
bispecific antibodies, they were only a minor population, and
extensive purification was required to isolate the desired
antibody. An engineering solution to this was the use of antibody
fragments to make bispecifics. Because such fragments lack the
complex quaternary structure of a full length antibody, variable
light and heavy chains can be linked in single genetic constructs.
Antibody fragments of many different forms have been generated,
including diabodies, single chain diabodies, tandem scFv's, and
Fab.sub.2 bispecifics (Chames & Baty, 2009, mAbs 1[6]:1-9;
Holliger & Hudson, 2005, Nature Biotechnology 23[9]:1126-1136;
expressly incorporated herein by reference). While these formats
can be expressed at high levels in bacteria and may have favorable
penetration benefits due to their small size, they clear rapidly in
vivo and can present manufacturing obstacles related to their
production and stability. A principal cause of these drawbacks is
that antibody fragments typically lack the constant region of the
antibody with its associated functional properties, including
larger size, high stability, and binding to various Fc receptors
and ligands that maintain long half-life in serum (i.e. the
neonatal Fc receptor FcRn) or serve as binding sites for
purification (i.e. protein A and protein G).
[0004] More recent work has attempted to address the shortcomings
of fragment-based bispecifics by engineering dual binding into full
length antibody-like formats (Wu et al., 2007, Nature Biotechnology
25[11]:1290-1297; U.S. Ser. No. 12/477,711; Michaelson et al.,
2009, mAbs 1[2]:128-141; PCT/US2008/074693; Zuo et al., 2000,
Protein Engineering 13[5]:361-367; U.S. Ser. No. 09/865,198; Shen
et al., 2006, J Biol Chem 281[16]:10706-10714; Lu et al., 2005, J
Biol Chem 280[20]:19665-19672; PCT/US2005/025472; expressly
incorporated herein by reference). These formats overcome some of
the obstacles of the antibody fragment bispecifics, principally
because they contain an Fc region. One significant drawback of
these formats is that, because they build new antigen binding sites
on top of the homodimeric constant chains, binding to the new
antigen is always bivalent.
[0005] For many antigens that are attractive as co-targets in a
therapeutic bispecific format, the desired binding is monovalent
rather than bivalent. For many immune receptors, cellular
activation is accomplished by cross-linking of a monovalent binding
interaction. The mechanism of cross-linking is typically mediated
by antibody/antigen immune complexes, or via effector cell to
target cell engagement. For example, the low affinity Fc gamma
receptors (Fc.gamma.Rs) such as Fc.gamma.RIIa, Fc.gamma.RIIb, and
Fc.gamma.RIIIa bind monovalently to the antibody Fc region.
Monovalent binding does not activate cells expressing these
Fc.gamma.Rs; however, upon immune complexation or cell-to-cell
contact, receptors are cross-linked and clustered on the cell
surface, leading to activation. For receptors responsible for
mediating cellular killing, for example Fc.gamma.RIIIa on natural
killer (NK) cells, receptor cross-linking and cellular activation
occurs when the effector cell engages the target cell in a highly
avid format (Bowles & Weiner, 2005, J Immunol Methods
304:88-99, expressly incorporated by reference). Similarly, on B
cells the inhibitory receptor Fc.gamma.RIIb downregulates B cell
activation only when it engages into an immune complex with the
cell surface B-cell receptor (BCR), a mechanism that is mediated by
immune complexation of soluble IgG's with the same antigen that is
recognized by the BCR (Heyman 2003, Immunol Lett 88[2]:157-161;
Smith and Clatworthy, 2010, Nature Reviews Immunology 10:328-343;
expressly incorporated by reference). As another example, CD3
activation of T-cells occurs only when its associated T-cell
receptor (TCR) engages antigen-loaded MHC on antigen presenting
cells in a highly avid cell-to-cell synapse (Kuhns et al., 2006,
Immunity 24:133-139). Indeed nonspecific bivalent cross-linking of
CD3 using an anti-CD3 antibody elicits a cytokine storm and
toxicity (Perruche et al., 2009, J Immunol 183[2]:953-61; Chatenoud
& Bluestone, 2007, Nature Reviews Immunology 7:622-632;
expressly incorporated by reference). Thus for practical clinical
use, the preferred mode of CD3 co-engagement for redirected killing
of targets cells is monovalent binding that results in activation
only upon engagement with the co-engaged target.
[0006] CD38, also known as cyclic ADP ribose hydrolase, is a type
II transmembrane glycoprotein with a long C-terminal extracellular
domain and a short N-terminal cytoplasmic domain. Among
hematopoietic cells, an assortment of functional effects have been
ascribed to CD38 mediated signaling, including lymphocyte
proliferation, cytokine release, regulation of B and myeloid cell
development and survival, and induction of dendritic cell
maturation. CD38 is unregulated in many hematopoeitic malignancies
and in cell lines derived from various hematopoietic malignancies
including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL),
multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B
and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL),
acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's
Lymphoma (HL), and chronic myeloid leukemia (CML). On the other
hand, most primitive pluripotent stem cells of the hematopoietic
system are CD38-. In spite of the recent progress in the discovery
and development of anti-cancer agents, many forms of cancer
involving CD38-expressing tumors still have a poor prognosis. Thus,
there is a need for improved methods for treating such forms of
cancer.
[0007] Thus while bispecifics generated from antibody fragments
suffer biophysical and pharmacokinetic hurdles, a drawback of those
built with full length antibody-like formats is that they engage
co-target antigens multivalently in the absence of the primary
target antigen, leading to nonspecific activation and potentially
toxicity. The present invention solves this problem by introducing
novel bispecific antibodies directed to CD3 and CD38.
BRIEF SUMMARY OF THE INVENTION
[0008] Accordingly, the present invention provides heterodimeric
antibodies directed against CD3 and CD38. In some embodiments, the
heterodimeric antibodies comprise a first monomer comprising SEQ ID
NO:91; a second monomer comprising SEQ ID NO:92; and a light chain
comprising SEQ ID NO:93. In some embodiments, the heterodimeric
antibodies comprise a first monomer comprising SEQ ID NO:88; a
second monomer comprising SEQ ID NO:89; and a light chain
comprising SEQ ID NO:90. The invention further provides nucleic
acid compositions comprising first, second and third nucleic acids
that encode the sequences above, as well as expression vectors
comprising the nucleic acid compositions, host cells comprising
either the nucleic acids or expression vectors, and methods of
making and using the heterodimeric antibodies.
[0009] In an additional aspect, the invention provides
heterodimeric antibodies comprising: a first monomer comprising: i)
a first Fc domain; ii) an anti-CD3 scFv comprising a scFv variable
light domain, an scFv linker and a scFv variable heavy domain;
wherein said scFv is covalently attached to the N-terminus of said
Fc domain using a domain linker; a second monomer comprising a
heavy chain comprising: i) a heavy variable domain; and ii) a heavy
chain constant domain comprising a second Fc domain; and a light
chain comprising a variable light domain and a variable light
constant domain. In some aspects the scFv variable light domain
comprises: a vlCDR1 having SEQ ID NO:15, a vlCDR2 having SEQ ID
NO:16 and a vlCDR3 having SEQ ID NO:17, said scFv variable heavy
domain comprises a vhCDR1 having SEQ ID NO:11, a vhCDR2 having SEQ
ID NO:12 and a vhCDR3 having SEQ ID NO:13, and wherein said heavy
variable domain and said variable light domain bind CD38.
[0010] In a further aspect, the invention provides heterodimeric
antibodies comprising: a) a first monomer comprising: i) a first Fc
domain; ii) an anti-CD3 scFv comprising a scFv variable light
domain, an scFv linker and a scFv variable heavy domain; wherein
said scFv is covalently attached to the N-terminus of said Fc
domain using a domain linker; b) a second monomer comprising a
heavy chain comprising: i) a heavy variable domain; and ii) a heavy
chain constant domain comprising a second Fc domain; and c) a light
chain comprising a variable light domain and a variable light
constant domain. In this aspect, the scFv variable light domain
comprises: a vlCDR1 having SEQ ID NO:24, a vlCDR2 having SEQ ID
NO:25 and a vlCDR3 having SEQ ID NO:26, said scFv variable heavy
domain comprises a vhCDR1 having SEQ ID NO:11, a vhCDR2 having SEQ
ID NO:12 and a vhCDR3 having SEQ ID NO:13, and wherein said heavy
variable domain and said variable light domain bind CD38.
[0011] In a further aspect, the invention provides heterodimeric
antibodies comprising: a) a first monomer comprising: i) a first Fc
domain; ii) an anti-CD3 scFv comprising a scFv variable light
domain, an scFv linker and a scFv variable heavy domain; wherein
said scFv is covalently attached to the N-terminus of said Fc
domain using a domain linker; b) a second monomer comprising a
heavy chain comprising: i) a heavy variable domain; and ii) a heavy
chain constant domain comprising a second Fc domain; and c) a light
chain comprising a variable light domain and a variable light
constant domain. In this aspect, the scFv variable light domain
comprises: a vlCDR1 having SEQ ID NO:33, a vlCDR2 having SEQ ID
NO:34 and a vlCDR3 having SEQ ID NO:35, said scFv variable heavy
domain comprises a vhCDR1 having SEQ ID NO:29, a vhCDR2 having SEQ
ID NO:30 and a vhCDR3 having SEQ ID NO:31, and wherein said heavy
variable domain and said variable light domain bind CD38.
[0012] In a further aspect, the invention provides heterodimeric
antibodies comprising: a) a first monomer comprising: i) a first Fc
domain; ii) an anti-CD3 scFv comprising a scFv variable light
domain, an scFv linker and a scFv variable heavy domain; wherein
said scFv is covalently attached to the N-terminus of said Fc
domain using a domain linker; b) a second monomer comprising a
heavy chain comprising: i) a heavy variable domain; and ii) a heavy
chain constant domain comprising a second Fc domain; and c) a light
chain comprising a variable light domain and a variable light
constant domain. In this aspect, the scFv variable light domain
comprises: a vlCDR1 having SEQ ID NO:42, a vlCDR2 having SEQ ID
NO:43 and a vlCDR3 having SEQ ID NO:44, said scFv variable heavy
domain comprises a vhCDR1 having SEQ ID NO:38, a vhCDR2 having SEQ
ID NO:39 and a vhCDR3 having SEQ ID NO:40, and wherein said heavy
variable domain and said variable light domain bind CD38.
[0013] In an additional aspect, the "bottle opener" heterodimeric
antibodies of the invention have a scFv that binds CD3 and vh and
vl domains, wherein the variable light domain comprises: a vlCDR1
having the sequence RASQNVDTWVA (SEQ ID NO:69), a vlCDR2 having the
sequence SASYRYS (SEQ ID NO:70) and a vlCDR3 having the sequence
QQYDSYPLT (SEQ ID NO:71), said variable heavy domain comprises a
vhCDR1 having the sequence RSWMN (SEQ ID NO:65), a vhCDR2 having
the sequence EINPDSSTINYATSVKG (SEQ ID NO:66) and a vhCDR3 having
the sequence YGNWFPY (SEQ ID NO:67).
[0014] In additional embodiments, the variable light domain
comprises: a vlCDR1 having the sequence RASQNVDTNVA (SEQ ID NO:78),
a vlCDR2 having the sequence SASYRYS (SEQ ID NO:79) and a vlCDR3
having the sequence QQYDSYPLT (SEQ ID NO:80), said variable heavy
domain comprises a vhCDR1 having the sequence RSWMN (SEQ ID NO:74),
a vhCDR2 having the sequence EINPDSSTINYATSVKG (SEQ ID NO:75) and a
vhCDR3 having the sequence YGNWFPY (SEQ ID NO:76).
[0015] In a further aspect, the invention provides heterodimeric
antibodies comprising: a) a first monomer comprising: i) a first
heavy chain comprising: 1) a first variable heavy domain; 2) a
first constant heavy chain comprising a first Fc domain; 3) a scFv
comprising a scFv variable light domain, an scFv linker and a scFv
variable heavy domain; wherein said scFv is covalently attached to
the C-terminus of said Fc domain using a domain linker; b) a second
monomer comprising a second heavy chain comprising a second
variable heavy domain and a second constant heavy chain comprising
a second Fc domain; and c) a common light chain comprising a
variable light domain and a constant light domain; wherein said
first and said second Fc domains have a set of amino acid
substitutions selected from the group consisting of
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L and
K370S:S364K/E357Q, and wherein said first variable heavy domain and
said variable light domain bind human CD38 (SEQ ID NO:131), said
second variable heavy domain and said variable light domain bind
human CD38 (SEQ ID NO:131), and said scFv binds human CD3 (SEQ ID
NO:129).
[0016] In a further aspect, the invention provides heterodimeric
antibodies comprising: a) a first monomer comprising: i) a first
heavy chain comprising: 1) a first variable heavy domain; 2) a
first constant heavy domain comprising a first Fc domain; and 3) a
first variable light domain, wherein said first variable light
domain is covalently attached to the C-terminus of said first Fc
domain using a domain linker; b) a second monomer comprising: i) a
second variable heavy domain; ii) a second constant heavy domain
comprising a second Fc domain; and iii) a third variable heavy
domain, wherein said second variable heavy domain is covalently
attached to the C-terminus of said second Fc domain using a domain
linker;
[0017] c) a common light chain comprising a variable light domain
and a constant light domain; wherein said first and said second Fc
domain have a set of amino acid substitutions selected from the
group consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L
and K370S:S364K/E357Q wherein said first variable heavy domain and
said variable light domain bind human CD38 (SEQ ID NO:131), said
second variable heavy domain and said variable light domain bind
said human CD38 (SEQ ID NO:131), and said second variable light
domain and said third variable heavy domain binds human CD3 (SEQ ID
NO:129).
[0018] In a further aspect, the invention provides heterodimeric
antibodies comprising: a) a first monomer comprising: i) a first
heavy chain comprising: 1) a first variable heavy domain; 2) a
first constant heavy chain comprising a first CH1 domain and a
first Fc domain; 3) a scFv comprising a scFv variable light domain,
an scFv linker and a scFv variable heavy domain; wherein said scFv
is covalently attached between the C-terminus of said CH1 domain
and the N-terminus of said first Fc domain using domain linkers; b)
a second monomer comprising a second heavy chain comprising a
second variable heavy domain and a second constant heavy chain
comprising a second Fc domain; and c) a common light chain
comprising a variable light domain and a constant light domain;
wherein said first and said second Fc domain have a set of amino
acid substitutions selected from the group consisting of
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L and
K370S:S364K/E357Q, wherein said first variable heavy domain and
said variable light domain bind human CD38 (SEQ ID NO:131), said
second variable heavy domain and said variable light domain bind
said human CD38 (SEQ ID NO:131), and said scFv binds human CD3 (SEQ
ID NO:129).
[0019] In a further aspect, the invention provides heterodimeric
antibodies comprising: a) a first monomer comprising: i) a first
heavy chain comprising: 1) a first variable heavy domain; 2) a
first constant heavy domain comprising a first Fc domain; and 3) a
first variable light domain, wherein said second variable light
domain is covalently attached between the C-terminus of the CH1
domain of said first constant heavy domain and the N-terminus of
said first Fc domain using domain linkers; b) a second monomer
comprising: i) a second variable heavy domain; ii) a second
constant heavy domain comprising a second Fc domain; and iii) a
third variable heavy domain, wherein said second variable heavy
domain is covalently attached to the C-terminus of said second Fc
domain using a domain linker; c) a common light chain comprising a
variable light domain and a constant light domain; wherein said
first and said second Fc domains have a set of amino acid
substitutions selected from the group consisting of
S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;
T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L and
K370S:S364K/E357Q wherein said first variable heavy domain and said
variable light domain bind human CD38 (SEQ ID NO:131), said second
variable heavy domain and said variable light domain bind said
human CD38 (SEQ ID NO:131), and said second variable light domain
and said third variable heavy domain binds human CD3 (SEQ ID
NO:129).
[0020] In an additional aspect, the invention provides
heterodimeric antibodies comprising a) a first monomer comprising:
i) a first heavy chain comprising: 1) a first variable heavy
domain; 2) a first constant heavy chain comprising a first CH1
domain and a first Fc domain; 3) a scFv comprising a scFv variable
light domain, an scFv linker and a scFv variable heavy domain;
wherein said scFv is covalently attached between the C-terminus of
said CH1 domain and the N-terminus of said first Fc domain using
domain linkers; b) a second monomer comprising a second Fc domain;
and c) a light chain comprising a variable light domain and a
constant light domain; wherein said first and said second Fc domain
have a set of amino acid substitutions selected from the group
consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L
and K370S:S364K/E357Q, wherein said first variable heavy domain and
said variable light domain bind human CD38 (SEQ ID NO:131), said
scFv binds human CD3 (SEQ ID NO:129).
[0021] In an additional aspect, in some embodiments the
heterodimeric antibodies comprise a first Fc domain and a second Fc
domain which comprise a set of variants selected from the group
consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L
and K370S:S364K/E357Q.
[0022] In further aspects the scFv comprise scFv linkers that are
charged linkers.
[0023] In additional aspects the heavy chain constant domain of the
heterodimeric antibodies outlined herein comprise the amino acid
substitutions N208D/Q295E/N384D/Q418E/N421D.
[0024] In a further aspect, the heterodimeric antibodies of the
invention have first and second Fc domains which comprise the amino
acid substitutions E233P/L234V/L235A/G236del/S267K.
[0025] In an additional aspect, the invention provides nucleic acid
composition encoding the heterodimeric antibodies of the invention
that comprises a) a first nucleic acid encoding said first monomer;
b) a second nucleic acid encoding said second monomer; and c) a
third nucleic acid encoding said light chain.
[0026] In a further aspect, the invention provides expression
vector compositions comprising: a) a first expression vector
comprising a nucleic acid encoding said first monomer; b) a second
expression vector comprising a nucleic acid encoding said second
monomer; and c) a third expression vector comprising a nucleic acid
encoding said light chain. The invention further provides host
cells comprising either the nucleic acid compositions or the
expression vector compositions.
[0027] The invention further provides methods of making the
heterodimeric antibodies comprising culturing the host cells under
conditions wherein said antibody is expressed, and recovering said
antibody.
[0028] The invention further provides methods of treating cancer
comprising administering a heterodimeric antibody of the invention
to a patient in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIGS. 1A and 1B depict several formats of the present
invention. Two forms of the "bottle opener" format are depicted,
one with the anti-CD3 antigen binding domain comprising a scFv and
the anti-CD38 antigen binding domain comprising a Fab, and one with
these reversed. The mAb-Fv, mAb-scFv, Central-scFv and Central-Fv
formats are all shown. In addition, "one-armed" formats, where one
monomer just comprises an Fc domain, are shown, both a one arm
Central-scFv and a one arm Central-Fv. A dual scFv format is also
shown.
[0030] FIG. 2 depicts the sequences of the "High CD3"
anti-CD3_H1.30_L1.47 construct, including the variable heavy and
light domains (CDRs underlined), as well as the individual vl and
vhCDRs, as well as an scFv construct with a charged linker (double
underlined). As is true of all the sequences depicted in the
Figures, this charged linker may be replaced by an uncharged linker
or a different charged linker, as needed.
[0031] FIG. 3 depicts the sequences of the "High-Int #1"
Anti-CD3_H1.32_L1.47 construct, including the variable heavy and
light domains (CDRs underlined), as well as the individual vl and
vhCDRs, as well as an scFv construct with a charged linker (double
underlined). As is true of all the sequences depicted in the
Figures, this charged linker may be replaced by an uncharged linker
or a different charged linker, as needed.
[0032] FIG. 4 depicts the sequences of the "High-Int #2"
Anti-CD3_H1.89_L1.47 construct, including the variable heavy and
light domains (CDRs underlined), as well as the individual vl and
vhCDRs, as well as an scFv construct with a charged linker (double
underlined). As is true of all the sequences depicted in the
Figures, this charged linker may be replaced by an uncharged linker
or a different charged linker, as needed.
[0033] FIG. 5 depicts the sequences of the "High-Int #3"
Anti-CD3_H1.90_L1.47 construct, including the variable heavy and
light domains (CDRs underlined), as well as the individual vl and
vhCDRs, as well as an scFv construct with a charged linker (double
underlined). As is true of all the sequences depicted in the
Figures, this charged linker may be replaced by an uncharged linker
or a different charged linker, as needed.
[0034] FIG. 6 depicts the sequences of the "Int"
Anti-CD3_H1.90_L1.47 construct, including the variable heavy and
light domains (CDRs underlined), as well as the individual vl and
vhCDRs, as well as an scFv construct with a charged linker (double
underlined). As is true of all the sequences depicted in the
Figures, this charged linker may be replaced by an uncharged linker
or a different charged linker, as needed.
[0035] FIG. 7 depicts the sequences of the "Low"
Anti-CD3_H1.31_L1.47 construct, including the variable heavy and
light domains (CDRs underlined), as well as the individual vl and
vhCDRs, as well as an scFv construct with a charged linker (double
underlined). As is true of all the sequences depicted in the
Figures, this charged linker may be replaced by an uncharged linker
or a different charged linker, as needed.
[0036] FIG. 8 depicts the sequences of the High CD38:
OKT10_H1.77_L1.24 construct, including the variable heavy and light
domains (CDRs underlined), as well as the individual vl and vhCDRs,
as well as an scFv construct with a charged linker (double
underlined).
[0037] FIG. 9 depicts the sequences of the intermediate CD38:
OKT10_H1L1.24 construct, including the variable heavy and light
domains (CDRs underlined), as well as the individual vl and vhCDRs,
as well as an scFv construct with a charged linker (double
underlined).
[0038] FIG. 10 depicts the sequences of the Low CD38: OKT10_H1L1
construct, including the variable heavy and light domains (CDRs
underlined), as well as the individual vl and vhCDRs, as well as an
scFv construct with a charged linker (double underlined).
[0039] FIG. 11 depicts the sequences of XENP15331.
[0040] FIG. 12 depicts the sequences of XENP13243.
[0041] FIG. 13 depicts the sequences of XENP14702.
[0042] FIG. 14 depicts the sequences of XENP15426.
[0043] FIG. 15 depicts the sequences of XENP14701.
[0044] FIG. 16 depicts the sequence of XENP14703.
[0045] FIG. 17 depicts the sequence of XENP13243.
[0046] FIG. 18 depicts the sequences of XENP18967.
[0047] FIG. 19 depicts the sequences of XENP18971.
[0048] FIG. 20 depicts the sequences of XENP18969.
[0049] FIG. 21 depicts the sequences of XENP18970.
[0050] FIG. 22 depicts the sequences of XENP18972.
[0051] FIG. 23 depicts the sequences of XENP18973.
[0052] FIG. 24 depicts the sequences of XENP15055.
[0053] FIG. 25 depicts the sequences of XENP13544.
[0054] FIG. 26 depicts the sequences of XENP13694.
[0055] FIG. 27 depicts the sequence of human CD3 .epsilon..
[0056] FIG. 28 depicts the full length (SEQ ID NO:130) and
extracellular domain (ECD; SEQ ID NO:131) of the human CD38
protein.
[0057] FIGS. 29A-29E depict useful pairs of heterodimerization
variant sets (including skew and pI variants).
[0058] FIG. 30 depict a list of isosteric variant antibody constant
regions and their respective substitutions. pI_(-) indicates lower
pI variants, while pI_(+) indicates higher pI variants. These can
be optionally and independently combined with other
heterodimerization variants of the invention (and other variant
types as well, as outlined herein).
[0059] FIG. 31 depict useful ablation variants that ablate
Fc.gamma.R binding (sometimes referred to as "knock outs" or "KO"
variants).
[0060] FIG. 32 show two particularly useful embodiments of the
invention.
[0061] FIGS. 33A and 33B depicts a number of charged scFv linkers
that find use in increasing or decreasing the pI of heterodimeric
antibodies that utilize one or more scFv as a component. A single
prior art scFv linker with a single charge is referenced as
"Whitlow", from Whitlow et al., Protein Engineering 6(8):989-995
(1993). It should be noted that this linker was used for reducing
aggregation and enhancing proteolytic stability in scFvs.
[0062] FIG. 34 depicts a list of engineered heterodimer-skewing Fc
variants with heterodimer yields (determined by HPLC-CIEX) and
thermal stabilities (determined by DSC). Not determined thermal
stability is denoted by "n.d.".
[0063] FIG. 35 Expression yields of bispecifics after protein A
affinity purification.
[0064] FIG. 36 Cationic exchange purification chromatograms.
[0065] FIG. 37 Redirected T cell cytotoxicity assay, 24 h
incubation, 10 k RPMI8226 cells, 400 k T cells. Test articles are
anti-CD38.times.anti-CD3 bispecifics. Detection was by LDH
[0066] FIG. 38 Redirected T cell cytotoxicity assay, 24 h
incubation, 10 k RPMI8226 cells, 500 k human PBMCs. Test articles
are anti-CD38.times.anti-CD3 bispecifics. Detection was by LDH.
[0067] FIG. 39 depicts the sequences of XENP14419,
[0068] FIG. 40 depicts the sequences of XENP14420.
[0069] FIG. 41 depicts the sequences of XENP14421.
[0070] FIG. 42 depicts the sequences of XENP14422.
[0071] FIG. 43 depicts the sequences of XENP14423.
[0072] FIG. 44 Redirected T cell cytotoxicity assay, 96 h
incubation, 40 k RPMI8226 cells, 400 k human PBMC. Test articles
are anti-CD38.times.anti-CD3 Fab-scFv-Fcs. Detection was by flow
cytometry, specifically the disappearance of CD38+ cells.
[0073] FIG. 45 Further analysis of redirected T cell cytotoxicity
assay described in FIG. 1. The first row shows the Mean
Fluorescence Intensity (MFI) of activation marker CD69 on CD4+ and
CD8+ T cells as detected by flow cytometry. The second row shows
the percentage of CD4+ and CD8+ T cells that are Ki-67+, a measure
of cell proliferation. The third row shows the intracellular Mean
Fluorescence Intensity (MFI) of granzyme B inhibitor PI-9 on CD4+
and CD8+ T cells as detected by flow cytometry.
[0074] FIG. 46 Design of mouse study to examine anti-tumor activity
of anti-CD38.times.anti-CD3 Fab-scFv-Fc bispecifics.
[0075] FIG. 47 Tumor size measured by IVIS.RTM. as a function of
time and treatment
[0076] FIG. 48 IVIS.RTM. bioluminescent images (Day 10)
[0077] FIG. 49 Depletion of CD38.sup.+ cells in cynomolgus monkeys
following single doses of the indicated test articles
[0078] FIG. 50 T cell activation measured by CD69 Mean Fluorescence
Intensity (MFI) in cynomolgus monkeys, color coding as in FIG.
49.
[0079] FIG. 51 Serum levels of IL-6, following single doses of the
indicated test articles.
[0080] FIG. 52 depicts the sequences of XENP15427.
[0081] FIG. 53 depicts the sequences of XENP15428.
[0082] FIG. 54 depicts the sequences of XENP15429.
[0083] FIG. 55 depicts the sequences of XENP15430.
[0084] FIG. 56 depicts the sequences of XENP15431.
[0085] FIG. 57 depicts the sequences of XENP15432.
[0086] FIG. 58 depicts the sequences of XENP15433.
[0087] FIG. 59 depicts the sequences of XENP15434.
[0088] FIG. 60 depicts the sequences of XENP15435.
[0089] FIG. 61 depicts the sequences of XENP15436.
[0090] FIG. 62 depicts the sequences of XENP15437.
[0091] FIG. 63 depicts the sequences of XENP15438.
[0092] FIG. 64 shows binding affinities in a Biacore assay.
[0093] FIG. 65 shows the Heterodimer purity during stable pool
generation using varied Light chain, Fab-Fc, and scFv-Fc
ratios.
[0094] FIG. 66 Human IgM and IgG2 depletion by
anti-CD38.times.anti-CD3 bispecifics in a huPBMC mouse model.
[0095] FIGS. 67A-67B depicts stability-optimized, humanized
anti-CD3 variant scFvs. Substitutions are given relative to the
H1_L1.4 scFv sequence. Amino acid numbering is Kabat numbering.
[0096] FIGS. 68A-68Z. Amino acid sequences of stability-optimized,
humanized anti-CD3 variant scFvs. CDRs are underlined. For each
heavy chain/light chain combination, four sequences are listed: (i)
scFv with C-terminal 6.times.His tag, (ii) scFv alone, (iii) VH
alone, (iv) VL alone.
[0097] FIG. 69 Redirected T cell cytotoxicity assay, 24 h
incubation, 10 k RPMI8226 cells, 500 k PBMC. Test articles are
anti-CD38 (OKT10_H1L1, OKT10_H1.77_L1.24).times.anti-CD3
Fab-scFv-Fcs. Detection was by LDH.
[0098] FIG. 70 huPBL-SCID Ig-depletion study. Test articles were
dosed 8 d after PBMC engraftment at 0.03, 0.3, or 3 mg/kg. Route of
administration was intraperitoneal. Blood samples were taken 14 d
after PBMC engraftment, processed to serum, and assayed for human
IgM and IgG2.
[0099] FIG. 71 depicts the sequences of XENP18967 Anti-CD38.
[0100] FIG. 72 depicts the sequences of XENP18971.
[0101] FIG. 73 depicts the sequences of XENP18969.
[0102] FIG. 74 depicts the sequences of. XENP18970.
[0103] FIG. 75 depicts the sequences of XENP18972.
[0104] FIG. 76 depicts the sequences of XENP18973.
[0105] FIG. 77 shows a matrix of possible combinations for
embodiments of the invention. An "A" means that the CDRs of the
referenced CD3 sequences can be combined with the CDRs of CD38
construct on the left hand side. That is, for example for the top
left hand cell, the vhCDRs from the variable heavy chain CD3 H1.30
sequence and the vlCDRs from the variable light chain of CD3 L1.47
sequence can be combined with the vhCDRs from the CD38 OKT10 H1.77
sequence and the vlCDRs from the OKT10L1.24 sequence. A "B" means
that the CDRs from the CD3 constructs can be combined with the
variable heavy and light domains from the CD38 construct. That is,
for example for the top left hand cell, the vhCDRs from the
variable heavy chain CD3 H1.30 sequence and the vlCDRs from the
variable light chain of CD3 L1.47 sequence can be combined with the
variable heavy domain CD38 OKT10 H1.77 sequence and the OKT10L1.24
sequence. A "C" is reversed, such that the variable heavy domain
and variable light domain from the CD3 sequences are used with the
CDRs of the CD38 sequences. A "D" is where both the variable heavy
and variable light chains from each are combined. An "E" is where
the scFv of the CD3 is used with the CDRs of the CD38 antigen
binding domain construct, and an "F" is where the scFv of the CD3
is used with the variable heavy and variable light domains of the
CD38 antigen binding domain.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0106] In order that the application may be more completely
understood, several definitions are set forth below. Such
definitions are meant to encompass grammatical equivalents.
[0107] By "ablation" herein is meant a decrease or removal of
activity. Thus for example, "ablating Fc.gamma.R binding" means the
Fc region amino acid variant has less than 50% starting binding as
compared to an Fc region not containing the specific variant, with
less than 70-80-90-95-98% loss of activity being preferred, and in
general, with the activity being below the level of detectable
binding in a Biacore assay. Of particular use in the ablation of
Fc.gamma.R binding are those shown in FIG. 16.
[0108] By "ADCC" or "antibody dependent cell-mediated cytotoxicity"
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. ADCC is correlated with binding to Fc.gamma.RIIIa;
increased binding to Fc.gamma.RIIIa leads to an increase in ADCC
activity.
[0109] By "ADCP" or antibody dependent cell-mediated phagocytosis
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause phagocytosis
of the target cell.
[0110] By "modification" herein is meant an amino acid
substitution, insertion, and/or deletion in a polypeptide sequence
or an alteration to a moiety chemically linked to a protein. For
example, a modification may be an altered carbohydrate or PEG
structure attached to a protein. By "amino acid modification"
herein is meant an amino acid substitution, insertion, and/or
deletion in a polypeptide sequence. For clarity, unless otherwise
noted, the amino acid modification is always to an amino acid coded
for by DNA, e.g. the 20 amino acids that have codons in DNA and
RNA.
[0111] By "amino acid substitution" or "substitution" herein is
meant the replacement of an amino acid at a particular position in
a parent polypeptide sequence with a different amino acid. In
particular, in some embodiments, the substitution is to an amino
acid that is not naturally occurring at the particular position,
either not naturally occurring within the organism or in any
organism. For example, the substitution E272Y refers to a variant
polypeptide, in this case an Fc variant, in which the glutamic acid
at position 272 is replaced with tyrosine. For clarity, a protein
which has been engineered to change the nucleic acid coding
sequence but not change the starting amino acid (for example
exchanging CGG (encoding arginine) to CGA (still encoding arginine)
to increase host organism expression levels) is not an "amino acid
substitution"; that is, despite the creation of a new gene encoding
the same protein, if the protein has the same amino acid at the
particular position that it started with, it is not an amino acid
substitution.
[0112] By "amino acid insertion" or "insertion" as used herein is
meant the addition of an amino acid sequence at a particular
position in a parent polypeptide sequence. For example, -233E or
233E designates an insertion of glutamic acid after position 233
and before position 234. Additionally, -233ADE or A233ADE
designates an insertion of AlaAspGlu after position 233 and before
position 234.
[0113] By "amino acid deletion" or "deletion" as used herein is
meant the removal of an amino acid sequence at a particular
position in a parent polypeptide sequence. For example, E233- or
E233# or E233( )designates a deletion of glutamic acid at position
233. Additionally, EDA233- or EDA233# designates a deletion of the
sequence GluAspAla that begins at position 233.
[0114] By "variant protein" or "protein variant", or "variant" as
used herein is meant a protein that differs from that of a parent
protein by virtue of at least one amino acid modification. Protein
variant may refer to the protein itself, a composition comprising
the protein, or the amino sequence that encodes it. Preferably, the
protein variant has at least one amino acid modification compared
to the parent protein, e.g. from about one to about seventy amino
acid modifications, and preferably from about one to about five
amino acid modifications compared to the parent. As described
below, in some embodiments the parent polypeptide, for example an
Fc parent polypeptide, is a human wild type sequence, such as the
Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequences
with variants can also serve as "parent polypeptides", for example
the IgG1/2 hybrid of FIG. 19. The protein variant sequence herein
will preferably possess at least about 80% identity with a parent
protein sequence, and most preferably at least about 90% identity,
more preferably at least about 95-98-99% identity. Variant protein
can refer to the variant protein itself, compositions comprising
the protein variant, or the DNA sequence that encodes it.
Accordingly, by "antibody variant" or "variant antibody" as used
herein is meant an antibody that differs from a parent antibody by
virtue of at least one amino acid modification, "IgG variant" or
"variant IgG" as used herein is meant an antibody that differs from
a parent IgG (again, in many cases, from a human IgG sequence) by
virtue of at least one amino acid modification, and "immunoglobulin
variant" or "variant immunoglobulin" as used herein is meant an
immunoglobulin sequence that differs from that of a parent
immunoglobulin sequence by virtue of at least one amino acid
modification. "Fc variant" or "variant Fc" as used herein is meant
a protein comprising an amino acid modification in an Fc domain.
The Fc variants of the present invention are defined according to
the amino acid modifications that compose them. Thus, for example,
N434S or 434S is an Fc variant with the substitution serine at
position 434 relative to the parent Fc polypeptide, wherein the
numbering is according to the EU index. Likewise, M428L/N434S
defines an Fc variant with the substitutions M428L and N434S
relative to the parent Fc polypeptide. The identity of the WT amino
acid may be unspecified, in which case the aforementioned variant
is referred to as 428L/434S. It is noted that the order in which
substitutions are provided is arbitrary, that is to say that, for
example, 428L/434S is the same Fc variant as M428L/N434S, and so
on. For all positions discussed in the present invention that
relate to antibodies, unless otherwise noted, amino acid position
numbering is according to the EU index. The EU index or EU index as
in Kabat or EU numbering scheme refers to the numbering of the EU
antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,
hereby entirely incorporated by reference.) The modification can be
an addition, deletion, or substitution. Substitutions can include
naturally occurring amino acids and, in some cases, synthetic amino
acids. Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO
03/073238; US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W.
Chin et al., (2002), Journal of the American Chemical Society
124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem
11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of
America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002),
Chem. 1-10, all entirely incorporated by reference.
[0115] As used herein, "protein" herein is meant at least two
covalently attached amino acids, which includes proteins,
polypeptides, oligopeptides and peptides. The peptidyl group may
comprise naturally occurring amino acids and peptide bonds, or
synthetic peptidomimetic structures, i.e. "analogs", such as
peptoids (see Simon et al., PNAS USA 89(20):9367 (1992), entirely
incorporated by reference). The amino acids may either be naturally
occurring or synthetic (e.g. not an amino acid that is coded for by
DNA); as will be appreciated by those in the art. For example,
homo-phenylalanine, citrulline, ornithine and noreleucine are
considered synthetic amino acids for the purposes of the invention,
and both D- and L-(R or S) configured amino acids may be utilized.
The variants of the present invention may comprise modifications
that include the use of synthetic amino acids incorporated using,
for example, the technologies developed by Schultz and colleagues,
including but not limited to methods described by Cropp &
Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004,
Proc Natl Acad Sci USA 101 (2):7566-71, Zhang et al., 2003,
303(5656):371-3, and Chin et al., 2003, Science 301(5635):964-7,
all entirely incorporated by reference. In addition, polypeptides
may include synthetic derivatization of one or more side chains or
termini, glycosylation, PEGylation, circular permutation,
cyclization, linkers to other molecules, fusion to proteins or
protein domains, and addition of peptide tags or labels.
[0116] By "residue" as used herein is meant a position in a protein
and its associated amino acid identity. For example, Asparagine 297
(also referred to as Asn297 or N297) is a residue at position 297
in the human antibody IgG1.
[0117] By "Fab" or "Fab region" as used herein is meant the
polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin
domains. Fab may refer to this region in isolation, or this region
in the context of a full length antibody, antibody fragment or Fab
fusion protein. By "Fv" or "Fv fragment" or "Fv region" as used
herein is meant a polypeptide that comprises the VL and VH domains
of a single antibody. As will be appreciated by those in the art,
these generally are made up of two chains.
[0118] By "IgG subclass modification" or "isotype modification" as
used herein is meant an amino acid modification that converts one
amino acid of one IgG isotype to the corresponding amino acid in a
different, aligned IgG isotype. For example, because IgG1 comprises
a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y
substitution in IgG2 is considered an IgG subclass
modification.
[0119] By "non-naturally occurring modification" as used herein is
meant an amino acid modification that is not isotypic. For example,
because none of the IgGs comprise a serine at position 434, the
substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof)
is considered a non-naturally occurring modification.
[0120] By "amino acid" and "amino acid identity" as used herein is
meant one of the 20 naturally occurring amino acids that are coded
for by DNA and RNA.
[0121] By "effector function" as used herein is meant a biochemical
event that results from the interaction of an antibody Fc region
with an Fc receptor or ligand. Effector functions include but are
not limited to ADCC, ADCP, and CDC.
[0122] By "IgG Fc ligand" as used herein is meant a molecule,
preferably a polypeptide, from any organism that binds to the Fc
region of an IgG antibody to form an Fc/Fc ligand complex. Fc
ligands include but are not limited to Fc.gamma.RIs, Fc.gamma.RIIs,
Fc.gamma.RIIIs, FcRn, C1q, C3, mannan binding lectin, mannose
receptor, staphylococcal protein A, streptococcal protein G, and
viral Fc.gamma.R. Fc ligands also include Fc receptor homologs
(FcRH), which are a family of Fc receptors that are homologous to
the Fc.gamma.Rs (Davis et al., 2002, Immunological Reviews
190:123-136, entirely incorporated by reference). Fc ligands may
include undiscovered molecules that bind Fc. Particular IgG Fc
ligands are FcRn and Fc gamma receptors. By "Fc ligand" as used
herein is meant a molecule, preferably a polypeptide, from any
organism that binds to the Fc region of an antibody to form an
Fc/Fc ligand complex.
[0123] By "Fc gamma receptor", "Fc.gamma.R" or "FcqammaR" as used
herein is meant any member of the family of proteins that bind the
IgG antibody Fc region and is encoded by an Fc.gamma.R gene. In
humans this family includes but is not limited to Fc.gamma.RI
(CD64), including isoforms Fc.gamma.RIa, Fc.gamma.RIb, and
Fc.gamma.RIc; Fc.gamma.RII (CD32), including isoforms Fc.gamma.RIIa
(including allotypes H131 and R131), Fc.gamma.RIIb (including
Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and
Fc.gamma.RIII (CD16), including isoforms Fc.gamma.RIIIa (including
allotypes V158 and F158) and Fc.gamma.RIIIb (including allotypes
Fc.gamma.RIIb-NA1 and Fc.gamma.RIIb-NA2) (Jefferis et al., 2002,
Immunol Lett 82:57-65, entirely incorporated by reference), as well
as any undiscovered human Fc.gamma.Rs or Fc.gamma.R isoforms or
allotypes. An Fc.gamma.R may be from any organism, including but
not limited to humans, mice, rats, rabbits, and monkeys. Mouse
Fc.gamma.Rs include but are not limited to Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and Fc.gamma.RIII-2
(CD16-2), as well as any undiscovered mouse Fc.gamma.Rs or
Fc.gamma.R isoforms or allotypes.
[0124] By "FcRn" or "neonatal Fc Receptor" as used herein is meant
a protein that binds the IgG antibody Fc region and is encoded at
least in part by an FcRn gene. The FcRn may be from any organism,
including but not limited to humans, mice, rats, rabbits, and
monkeys. As is known in the art, the functional FcRn protein
comprises two polypeptides, often referred to as the heavy chain
and light chain. The light chain is beta-2-microglobulin and the
heavy chain is encoded by the FcRn gene. Unless otherwise noted
herein, FcRn or an FcRn protein refers to the complex of FcRn heavy
chain with beta-2-microglobulin. A variety of FcRn variants used to
increase binding to the FcRn receptor, and in some cases, to
increase serum half-life, are shown in the Figure Legend of FIG.
83.
[0125] By "parent polypeptide" as used herein is meant a starting
polypeptide that is subsequently modified to generate a variant.
The parent polypeptide may be a naturally occurring polypeptide, or
a variant or engineered version of a naturally occurring
polypeptide. Parent polypeptide may refer to the polypeptide
itself, compositions that comprise the parent polypeptide, or the
amino acid sequence that encodes it. Accordingly, by "parent
immunoglobulin" as used herein is meant an unmodified
immunoglobulin polypeptide that is modified to generate a variant,
and by "parent antibody" as used herein is meant an unmodified
antibody that is modified to generate a variant antibody. It should
be noted that "parent antibody" includes known commercial,
recombinantly produced antibodies as outlined below.
[0126] By "Fc" or "Fc region" or "Fc domain" as used herein is
meant the polypeptide comprising the constant region of an antibody
excluding the first constant region immunoglobulin domain and in
some cases, part of the hinge. Thus Fc refers to the last two
constant region immunoglobulin domains of IgA, IgD, and IgG, the
last three constant region immunoglobulin domains of IgE and IgM,
and the flexible hinge N-terminal to these domains. For IgA and
IgM, Fc may include the J chain. For IgG, the Fc domain comprises
immunoglobulin domains C.gamma.2 and C.gamma.3 (C.gamma.2 and
C.gamma.3) and the lower hinge region between C.gamma.1 (C.gamma.1)
and C.gamma.2 (C.gamma.2). Although the boundaries of the Fc region
may vary, the human IgG heavy chain Fc region is usually defined to
include residues C226 or P230 to its carboxyl-terminus, wherein the
numbering is according to the EU index as in Kabat. In some
embodiments, as is more fully described below, amino acid
modifications are made to the Fc region, for example to alter
binding to one or more Fc.gamma.R receptors or to the FcRn
receptor.
[0127] By "heavy constant region" herein is meant the
CH1-hinge-CH2-CH3 portion of an antibody.
[0128] By "Fc fusion protein" or "immunoadhesin" herein is meant a
protein comprising an Fc region, generally linked (optionally
through a linker moiety, as described herein) to a different
protein, such as a binding moiety to a target protein, as described
herein. In some cases, one monomer of the heterodimeric antibody
comprises an antibody heavy chain (either including an scFv or
further including a light chain) and the other monomer is a Fc
fusion, comprising a variant Fc domain and a ligand. In some
embodiments, these "half antibody-half fusion proteins" are
referred to as "Fusionbodies".
[0129] By "position" as used herein is meant a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example the EU index for
antibody numbering.
[0130] By "target antigen" as used herein is meant the molecule
that is bound specifically by the variable region of a given
antibody. A target antigen may be a protein, carbohydrate, lipid,
or other chemical compound. A wide number of suitable target
antigens are described below.
[0131] By "strandedness" in the context of the monomers of the
heterodimeric antibodies of the invention herein is meant that,
similar to the two strands of DNA that "match", heterodimerization
variants are incorporated into each monomer so as to preserve the
ability to "match" to form heterodimers. For example, if some pI
variants are engineered into monomer A (e.g. making the pI higher)
then steric variants that are "charge pairs" that can be utilized
as well do not interfere with the pI variants, e.g. the charge
variants that make a pI higher are put on the same "strand" or
"monomer" to preserve both functionalities. Similarly, for "skew"
variants that come in pairs of a set as more fully outlined below,
the skilled artisan will consider pI in deciding into which strand
or monomer that incorporates one set of the pair will go, such that
pI separation is maximized using the pI of the skews as well.
[0132] By "target cell" as used herein is meant a cell that
expresses a target antigen.
[0133] By "variable region" as used herein is meant the region of
an immunoglobulin that comprises one or more Ig domains
substantially encoded by any of the V.kappa., V.lamda., and/or VH
genes that make up the kappa, lambda, and heavy chain
immunoglobulin genetic loci respectively.
[0134] By "wild type or WT" herein is meant an amino acid sequence
or a nucleotide sequence that is found in nature, including allelic
variations. A WT protein has an amino acid sequence or a nucleotide
sequence that has not been intentionally modified.
[0135] The antibodies of the present invention are generally
isolated or recombinant. "Isolated," when used to describe the
various polypeptides disclosed herein, means a polypeptide that has
been identified and separated and/or recovered from a cell or cell
culture from which it was expressed. Ordinarily, an isolated
polypeptide will be prepared by at least one purification step. An
"isolated antibody," refers to an antibody which is substantially
free of other antibodies having different antigenic specificities.
"Recombinant" means the antibodies are generated using recombinant
nucleic acid techniques in exogeneous host cells.
[0136] "Specific binding" or "specifically binds to" or is
"specific for" a particular antigen or an epitope means binding
that is measurably different from a non-specific interaction.
Specific binding can be measured, for example, by determining
binding of a molecule compared to binding of a control molecule,
which generally is a molecule of similar structure that does not
have binding activity. For example, specific binding can be
determined by competition with a control molecule that is similar
to the target.
[0137] Specific binding for a particular antigen or an epitope can
be exhibited, for example, by an antibody having a KD for an
antigen or epitope of at least about 10-4 M, at least about 10-5 M,
at least about 10-6 M, at least about 10-7 M, at least about 10-8
M, at least about 10-9 M, alternatively at least about 10-10 M, at
least about 10-11 M, at least about 10-12 M, or greater, where KD
refers to a dissociation rate of a particular antibody-antigen
interaction. Typically, an antibody that specifically binds an
antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-,
10,000- or more times greater for a control molecule relative to
the antigen or epitope.
[0138] Also, specific binding for a particular antigen or an
epitope can be exhibited, for example, by an antibody having a KA
or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-,
1000-, 5,000-, 10,000- or more times greater for the epitope
relative to a control, where KA or Ka refers to an association rate
of a particular antibody-antigen interaction.
II. Overview
[0139] Bispecific antibodies that co-engage CD3 and a tumor antigen
target have been designed and used to redirect T cells to attack
and lyse targeted tumor cells. Examples include the BiTE and DART
formats, which monovalently engage CD3 and a tumor antigen. While
the CD3-targeting approach has shown considerable promise, a common
side effect of such therapies is the associated production of
cytokines, often leading to toxic cytokine release syndrome.
Because the anti-CD3 binding domain of the bispecific antibody
engages all T cells, the high cytokine-producing CD4 T cell subset
is recruited. Moreover, the CD4 T cell subset includes regulatory T
cells, whose recruitment and expansion can potentially lead to
immune suppression and have a negative impact on long-term tumor
suppression. In addition, these formats do not contain Fc domains
and show very short serum half-lives in patients.
[0140] While the CD3-targeting approach has shown considerable
promise, a common side effect of such therapies is the associated
production of cytokines, often leading to toxic cytokine release
syndrome. Because the anti-CD3 binding domain of the bispecific
antibody engages all T cells, the high cytokine-producing CD4 T
cell subset is recruited. Moreover, the CD4 T cell subset includes
regulatory T cells, whose recruitment and expansion can potentially
lead to immune suppression and have a negative impact on long-term
tumor suppression. One such possible way to reduce cytokine
production and possibly reduce the activation of CD4 T cells is by
reducing the affinity of the anti-CD3 domain for CD3.
[0141] Accordingly, in some embodiments the present invention
provides antibody constructs comprising anti-CD3 antigen binding
domains that are "strong" or "high affinity" binders to CD3 (e.g.
one example are heavy and light variable domains depicted as
H1.30_L1.47 (optionally including a charged linker as appropriate))
and also bind to CD38. In other embodiments, the present invention
provides antibody constructs comprising anti-CD3 antigen binding
domains that are "lite" or "lower affinity" binders to CD3.
Additional embodiments provides antibody constructs comprising
anti-CD3 antigen binding domains that have intermediate or "medium"
affinity to CD3 that also bind to CD38.
[0142] It should be appreciated that the "high, medium, low"
anti-CD3 sequences of the present invention can be used in a
variety of heterodimerization formats. While the majority of the
disclosure herein uses the "bottle opener" format of heterodimers,
these variable heavy and light sequences, as well as the scFv
sequences (and Fab sequences comprising these variable heavy and
light sequences) can be used in other formats, such as those
depicted in FIG. 2 of WO Publication No. 2014/145806, the Figures,
formats and legend of which is expressly incorporated herein by
reference.
[0143] Accordingly, the present invention provides heterodimeric
antibodies that bind to two different antigens, e.g the antibodies
are "bispecific", in that they bind two different target antigens,
e.g. CD3 and CD38 in the present invention. These heterodimeric
antibodies can bind these target antigens either monovalently (e.g.
there is a single antigen binding domain such as a variable heavy
and variable light domain pair) or bivalently (there are two
antigen binding domains that each independently bind the antigen).
The heterodimeric antibodies of the invention are based on the use
different monomers which contain amino acid substitutions that
"skew" formation of heterodimers over homodimers, as is more fully
outlined below, coupled with "pI variants" that allow simple
purification of the heterodimers away from the homodimers, as is
similarly outlined below. For the heterodimeric bispecific
antibodies of the invention, the present invention generally relies
on the use of engineered or variant Fc domains that can
self-assemble in production cells to produce heterodimeric
proteins, and methods to generate and purify such heterodimeric
proteins.
III. Antibodies
[0144] The present invention relates to the generation of
bispecific antibodies that bind CD3 and CD38, generally therapeutic
antibodies. As is discussed below, the term "antibody" is used
generally. Antibodies that find use in the present invention can
take on a number of formats as described herein, including
traditional antibodies as well as antibody derivatives, fragments
and mimetics, described herein.
[0145] Traditional antibody structural units typically comprise a
tetramer. Each tetramer is typically composed of two identical
pairs of polypeptide chains, each pair having one "light"
(typically having a molecular weight of about 25 kDa) and one
"heavy" chain (typically having a molecular weight of about 50-70
kDa). Human light chains are classified as kappa and lambda light
chains. The present invention is directed to the IgG class, which
has several subclasses, including, but not limited to IgG1, IgG2,
IgG3, and IgG4. Thus, "isotype" as used herein is meant any of the
subclasses of immunoglobulins defined by the chemical and antigenic
characteristics of their constant regions. It should be understood
that therapeutic antibodies can also comprise hybrids of isotypes
and/or subclasses. For example, as shown in US Publication
2009/0163699, incorporated by reference, the present invention
covers pI engineering of IgG1/G2 hybrids.
[0146] The amino-terminal portion of each chain includes a variable
region of about 100 to 110 or more amino acids primarily
responsible for antigen recognition, generally referred to in the
art and herein as the "Fv domain" or "Fv region". In the variable
region, three loops are gathered for each of the V domains of the
heavy chain and light chain to form an antigen-binding site. Each
of the loops is referred to as a complementarity-determining region
(hereinafter referred to as a "CDR"), in which the variation in the
amino acid sequence is most significant. "Variable" refers to the
fact that certain segments of the variable region differ
extensively in sequence among antibodies. Variability within the
variable region is not evenly distributed. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-15 amino
acids long or longer.
[0147] Each VH and VL is composed of three hypervariable regions
("complementary determining regions," "CDRs") and four FRs,
arranged from amino-terminus to carboxy-terminus in the following
order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
[0148] The hypervariable region generally encompasses amino acid
residues from about amino acid residues 24-34 (LCDR1; "L" denotes
light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain
variable region and around about 31-35B (HCDR1; "H" denotes heavy
chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain
variable region; Kabat et al., SEQUENCES OF PROTEINS OF
IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991) and/or those residues
forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52
(LCDR2) and 91-96 (LCDR3) in the light chain variable region and
26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain
variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.
Specific CDRs of the invention are described below.
[0149] Throughout the present specification, the Kabat numbering
system is generally used when referring to a residue in the
variable domain (approximately, residues 1-107 of the light chain
variable region and residues 1-113 of the heavy chain variable
region) and the EU numbering system for Fc regions (e.g, Kabat et
al., supra (1991)).
[0150] The present invention provides a large number of different
CDR sets. In this case, a "full CDR set" comprises the three
variable light and three variable heavy CDRs, e.g. a vlCDR1,
vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. These can be part of a
larger variable light or variable heavy domain, respectfully. In
addition, as more fully outlined herein, the variable heavy and
variable light domains can be on separate polypeptide chains, when
a heavy and light chain is used (for example when Fabs are used),
or on a single polypeptide chain in the case of scFv sequences.
[0151] The CDRs contribute to the formation of the antigen-binding,
or more specifically, epitope binding site of antibodies. "Epitope"
refers to a determinant that interacts with a specific antigen
binding site in the variable region of an antibody molecule known
as a paratope. Epitopes are groupings of molecules such as amino
acids or sugar side chains and usually have specific structural
characteristics, as well as specific charge characteristics. A
single antigen may have more than one epitope.
[0152] The epitope may comprise amino acid residues directly
involved in the binding (also called immunodominant component of
the epitope) and other amino acid residues, which are not directly
involved in the binding, such as amino acid residues which are
effectively blocked by the specifically antigen binding peptide; in
other words, the amino acid residue is within the footprint of the
specifically antigen binding peptide.
[0153] Epitopes may be either conformational or linear. A
conformational epitope is produced by spatially juxtaposed amino
acids from different segments of the linear polypeptide chain. A
linear epitope is one produced by adjacent amino acid residues in a
polypeptide chain. Conformational and nonconformational epitopes
may be distinguished in that the binding to the former but not the
latter is lost in the presence of denaturing solvents.
[0154] An epitope typically includes at least 3, and more usually,
at least 5 or 8-10 amino acids in a unique spatial conformation.
Antibodies that recognize the same epitope can be verified in a
simple immunoassay showing the ability of one antibody to block the
binding of another antibody to a target antigen, for example
"binning."
[0155] The carboxy-terminal portion of each chain defines a
constant region primarily responsible for effector function. Kabat
et al. collected numerous primary sequences of the variable regions
of heavy chains and light chains. Based on the degree of
conservation of the sequences, they classified individual primary
sequences into the CDR and the framework and made a list thereof
(see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH
publication, No. 91-3242, E. A. Kabat et al., entirely incorporated
by reference).
[0156] In the IgG subclass of immunoglobulins, there are several
immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig)
domain" herein is meant a region of an immunoglobulin having a
distinct tertiary structure. Of interest in the present invention
are the heavy chain domains, including, the constant heavy (CH)
domains and the hinge domains. In the context of IgG antibodies,
the IgG isotypes each have three CH regions. Accordingly, "CH"
domains in the context of IgG are as follows: "CH1" refers to
positions 118-220 according to the EU index as in Kabat. "CH2"
refers to positions 237-340 according to the EU index as in Kabat,
and "CH3" refers to positions 341-447 according to the EU index as
in Kabat. As shown herein and described below, the pI variants can
be in one or more of the CH regions, as well as the hinge region,
discussed below.
[0157] It should be noted that the sequences depicted herein start
at the CH1 region, position 118; the variable regions are not
included except as noted. For example, the first amino acid of SEQ
ID NO: 2, while designated as position"1" in the sequence listing,
corresponds to position 118 of the CH1 region, according to EU
numbering.
[0158] Another type of Ig domain of the heavy chain is the hinge
region. By "hinge" or "hinge region" or "antibody hinge region" or
"immunoglobulin hinge region" herein is meant the flexible
polypeptide comprising the amino acids between the first and second
constant domains of an antibody. Structurally, the IgG CH1 domain
ends at EU position 220, and the IgG CH2 domain begins at residue
EU position 237. Thus for IgG the antibody hinge is herein defined
to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1),
wherein the numbering is according to the EU index as in Kabat. In
some embodiments, for example in the context of an Fc region, the
lower hinge is included, with the "lower hinge" generally referring
to positions 226 or 230. As noted herein, pI variants can be made
in the hinge region as well.
[0159] The light chain generally comprises two domains, the
variable light domain (containing the light chain CDRs and together
with the variable heavy domains forming the Fv region), and a
constant light chain region (often referred to as CL or
C.kappa.).
[0160] Another region of interest for additional substitutions,
outlined below, is the Fc region.
[0161] Thus, the present invention provides different antibody
domains. As described herein and known in the art, the
heterodimeric antibodies of the invention comprise different
domains within the heavy and light chains, which can be overlapping
as well. These domains include, but are not limited to, the Fc
domain, the CH1 domain, the CH2 domain, the CH3 domain, the hinge
domain, the heavy constant domain (CH1-hinge-Fc domain or
CH1-hinge-CH2-CH3), the variable heavy domain, the variable light
domain, the light constant domain, FAb domains and scFv
domains.
[0162] Thus, the "Fc domain" includes the -CH2-CH3 domain, and
optionally a hinge domain. The heavy chain comprises a variable
heavy domain and a constant domain, which includes a CH1-optional
hinge-Fc domain comprising a CH2-CH3. The light chain comprises a
variable light chain and the light constant domain.
[0163] Some embodiments of the invention comprise at least one scFv
domain, which, while not naturally occurring, generally includes a
variable heavy domain and a variable light domain, linked together
by a scFv linker. As shown herein, there are a number of suitable
scFv linkers that can be used, including traditional peptide bonds,
generated by recombinant techniques.
[0164] The linker peptide may predominantly include the following
amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide
should have a length that is adequate to link two molecules in such
a way that they assume the correct conformation relative to one
another so that they retain the desired activity. In one
embodiment, the linker is from about 1 to 50 amino acids in length,
preferably about 1 to 30 amino acids in length. In one embodiment,
linkers of 1 to 20 amino acids in length may be used, with from
about 5 to about 10 amino acids finding use in some embodiments.
Useful linkers include glycine-serine polymers, including for
example (GS)n, (GSGGS)n (SEQ ID NO:332), (GGGGS)n (SEQ ID NO:333),
and (GGGS)n (SEQ ID NO:334), where n is an integer of at least one
(and generally from 3 to 4), glycine-alanine polymers,
alanine-serine polymers, and other flexible linkers. Alternatively,
a variety of nonproteinaceous polymers, including but not limited
to polyethylene glycol (PEG), polypropylene glycol,
polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol, may find use as linkers, that is may find use
as linkers.
[0165] Other linker sequences may include any sequence of any
length of CL/CH1 domain but not all residues of CL/CH1 domain; for
example the first 5-12 amino acid residues of the CL/CH1 domains.
Linkers can be derived from immunoglobulin light chain, for example
C.kappa. or C.lamda.. Linkers can be derived from immunoglobulin
heavy chains of any isotype, including for example C.gamma.1,
C.gamma.2, C.gamma.3, C.gamma.4, C.alpha.1, C.alpha.2, C.delta.,
C.alpha., and C.mu.. Linker sequences may also be derived from
other proteins such as Ig-like proteins (e.g. TCR, FcR, KIR), hinge
region-derived sequences, and other natural sequences from other
proteins.
[0166] In some embodiments, the linker is a "domain linker", used
to link any two domains as outlined herein together. While any
suitable linker can be used, many embodiments utilize a
glycine-serine polymer, including for example (GS)n, (GSGGS)n (SEQ
ID NO:332), (GGGGS)n (SEQ ID NO:333), and (GGGS)n (SEQ ID NO:334),
where n is an integer of at least one (and generally from 3 to 4 to
5) as well as any peptide sequence that allows for recombinant
attachment of the two domains with sufficient length and
flexibility to allow each domain to retain its biological function.
In some cases, and with attention being paid to "strandedness", as
outlined below, charged domain linkers, as used in some embodiments
of scFv linkers can be used.
[0167] In some embodiments, the scFv linker is a charged scFv
linker, a number of which are shown in FIG. 33. Accordingly, the
present invention further provides charged scFv linkers, to
facilitate the separation in pI between a first and a second
monomer. That is, by incorporating a charged scFv linker, either
positive or negative (or both, in the case of scaffolds that use
scFvs on different monomers), this allows the monomer comprising
the charged linker to alter the pI without making further changes
in the Fc domains. These charged linkers can be substituted into
any scFv containing standard linkers. Again, as will be appreciated
by those in the art, charged scFv linkers are used on the correct
"strand" or monomer, according to the desired changes in pI. For
example, as discussed herein, to make triple F format heterodimeric
antibody, the original pI of the Fv region for each of the desired
antigen binding domains are calculated, and one is chosen to make
an scFv, and depending on the pI, either positive or negative
linkers are chosen.
[0168] Charged domain linkers can also be used to increase the pI
separation of the monomers of the invention as well, and thus those
included in FIG. 33 an be used in any embodiment herein where a
linker is utilized.
[0169] In some embodiments, the antibodies are full length. By
"full length antibody" herein is meant the structure that
constitutes the natural biological form of an antibody, including
variable and constant regions, including one or more modifications
as outlined herein, particularly in the Fc domains to allow either
heterodimerization formation or the purification of heterodimers
away from homodimers. Full length antibodies generally include Fab
and Fc domains, and can additionally contain extra antigen binding
domains such as scFvs, as is generally depicted in the Figures.
[0170] In one embodiment, the antibody is an antibody fragment, as
long as it contains at least one constant domain which can be
engineered to produce heterodimers, such as pI engineering. Other
antibody fragments that can be used include fragments that contain
one or more of the CH1, CH2, CH3, hinge and CL domains of the
invention that have been pI engineered. For example, Fc fusions are
fusions of the Fc region (CH2 and CH3, optionally with the hinge
region) fused to another protein. A number of Fc fusions are known
the art and can be improved by the addition of the
heterodimerization variants of the invention. In the present case,
antibody fusions can be made comprising CH1; CH1, CH2 and CH3; CH2;
CH3; CH2 and CH3; CH1 and CH3, any or all of which can be made
optionally with the hinge region, utilizing any combination of
heterodimerization variants described herein.
[0171] In particular, the formats depicted in FIG. 1 are
antibodies, usually referred to as "heterodimeric antibodies",
meaning that the protein has at least two associated Fc sequences
self-assembled into a heterodimeric Fc domain.
Chimeric and Humanized Antibodies
[0172] In some embodiments, the antibody can be a mixture from
different species, e.g. a chimeric antibody and/or a humanized
antibody. In general, both "chimeric antibodies" and "humanized
antibodies" refer to antibodies that combine regions from more than
one species. For example, "chimeric antibodies" traditionally
comprise variable region(s) from a mouse (or rat, in some cases)
and the constant region(s) from a human. "Humanized antibodies"
generally refer to non-human antibodies that have had the
variable-domain framework regions swapped for sequences found in
human antibodies. Generally, in a humanized antibody, the entire
antibody, except the CDRs, is encoded by a polynucleotide of human
origin or is identical to such an antibody except within its CDRs.
The CDRs, some or all of which are encoded by nucleic acids
originating in a non-human organism, are grafted into the
beta-sheet framework of a human antibody variable region to create
an antibody, the specificity of which is determined by the
engrafted CDRs. The creation of such antibodies is described in,
e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et
al., 1988, Science 239:1534-1536, all entirely incorporated by
reference. "Backmutation" of selected acceptor framework residues
to the corresponding donor residues is often required to regain
affinity that is lost in the initial grafted construct (U.S. Pat.
No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761;
U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No.
5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297; U.S.
Pat. No. 6,407,213, all entirely incorporated by reference). The
humanized antibody optimally also will comprise at least a portion
of an immunoglobulin constant region, typically that of a human
immunoglobulin, and thus will typically comprise a human Fc region.
Humanized antibodies can also be generated using mice with a
genetically engineered immune system. Roque et al., 2004,
Biotechnol. Prog. 20:639-654, entirely incorporated by reference. A
variety of techniques and methods for humanizing and reshaping
non-human antibodies are well known in the art (See Tsurushita
& Vasquez, 2004, Humanization of Monoclonal Antibodies,
Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and
references cited therein, all entirely incorporated by reference).
Humanization methods include but are not limited to methods
described in Jones et at, 1986, Nature 321:522-525; Riechmann et
al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science,
239:1534-1536; Queen et at, 1989, Proc Natl Acad Sci, USA
86:10029-33; He et at, 1998, J. Immunol. 160: 1029-1035; Carter et
at, 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et at, 1997,
Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad.
Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8,
all entirely incorporated by reference. Humanization or other
methods of reducing the immunogenicity of nonhuman antibody
variable regions may include resurfacing methods, as described for
example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA
91:969-973, entirely incorporated by reference. In one embodiment,
the parent antibody has been affinity matured, as is known in the
art. Structure-based methods may be employed for humanization and
affinity maturation, for example as described in U.S. Ser. No.
11/004,590. Selection based methods may be employed to humanize
and/or affinity mature antibody variable regions, including but not
limited to methods described in Wu et al., 1999, J. Mol. Biol.
294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;
Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et
al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al.,
2003, Protein Engineering 16(10):753-759, all entirely incorporated
by reference. Other humanization methods may involve the grafting
of only parts of the CDRs, including but not limited to methods
described in U.S. Ser. No. 09/810,510; Tan et al., 2002, J.
Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol.
169:3076-3084, all entirely incorporated by reference.
IV. Heterodimeric Antibodies
[0173] Accordingly, in some embodiments the present invention
provides heterodimeric antibodies that rely on the use of two
different heavy chain variant Fc domains that will self-assemble to
form heterodimeric antibodies.
[0174] The present invention is directed to novel constructs to
provide heterodimeric antibodies that allow binding to more than
one antigen or ligand, e.g. to allow for bispecific binding. The
heterodimeric antibody constructs are based on the self-assembling
nature of the two Fc domains of the heavy chains of antibodies,
e.g. two "monomers" that assemble into a "dimer". Heterodimeric
antibodies are made by altering the amino acid sequence of each
monomer as more fully discussed below. Thus, the present invention
is generally directed to the creation of heterodimeric antibodies
which can co-engage antigens in several ways, relying on amino acid
variants in the constant regions that are different on each chain
to promote heterodimeric formation and/or allow for ease of
purification of heterodimers over the homodimers.
[0175] Thus, the present invention provides bispecific antibodies.
An ongoing problem in antibody technologies is the desire for
"bispecific" antibodies that bind to two different antigens
simultaneously, in general thus allowing the different antigens to
be brought into proximity and resulting in new functionalities and
new therapies. In general, these antibodies are made by including
genes for each heavy and light chain into the host cells. This
generally results in the formation of the desired heterodimer
(A-B), as well as the two homodimers (A-A and B-B (not including
the light chain heterodimeric issues)). However, a major obstacle
in the formation of bispecific antibodies is the difficulty in
purifying the heterodimeric antibodies away from the homodimeric
antibodies and/or biasing the formation of the heterodimer over the
formation of the homodimers.
[0176] There are a number of mechanisms that can be used to
generate the heterodimers of the present invention. In addition, as
will be appreciated by those in the art, these mechanisms can be
combined to ensure high heterodimerization. Thus, amino acid
variants that lead to the production of heterodimers are referred
to as "heterodimerization variants". As discussed below,
heterodimerization variants can include steric variants (e.g. the
"knobs and holes" or "skew" variants described below and the
"charge pairs" variants described below) as well as "pI variants",
which allows purification of homodimers away from heterodimers. As
is generally described in WO2014/145806, hereby incorporated by
reference in its entirety and specifically as below for the
discussion of "heterodimerization variants", useful mechanisms for
heterodimerization include "knobs and holes" ("KIH"; sometimes
herein as "skew" variants (see discussion in WO2014/145806),
"electrostatic steering" or "charge pairs" as described in
WO2014/145806, pI variants as described in WO2014/145806, and
general additional Fc variants as outlined in WO2014/145806 and
below.
[0177] In the present invention, there are several basic mechanisms
that can lead to ease of purifying heterodimeric antibodies; one
relies on the use of pI variants, such that each monomer has a
different pI, thus allowing the isoelectric purification of A-A,
A-B and B-B dimeric proteins. Alternatively, some scaffold formats,
such as the "triple F" format, also allows separation on the basis
of size. As is further outlined below, it is also possible to
"skew" the formation of heterodimers over homodimers. Thus, a
combination of steric heterodimerization variants and pI or charge
pair variants find particular use in the invention.
[0178] In general, embodiments of particular use in the present
invention rely on sets of variants that include skew variants, that
encourage heterodimerization formation over homodimerization
formation, coupled with pI variants, which increase the pI
difference between the two monomers.
[0179] Additionally, as more fully outlined below, depending on the
format of the heterodimer antibody, pI variants can be either
contained within the constant and/or Fc domains of a monomer, or
charged linkers, either domain linkers or scFv linkers, can be
used. That is, scaffolds that utilize scFv(s) such as the Triple F
format can include charged scFv linkers (either positive or
negative), that give a further pI boost for purification purposes.
As will be appreciated by those in the art, some Triple F formats
are useful with just charged scFv linkers and no additional pI
adjustments, although the invention does provide pI variants that
are on one or both of the monomers, and/or charged domain linkers
as well. In addition, additional amino acid engineering for
alternative functionalities may also confer pI changes, such as Fc,
FcRn and KO variants.
[0180] In the present invention that utilizes pI as a separation
mechanism to allow the purification of heterodimeric proteins,
amino acid variants can be introduced into one or both of the
monomer polypeptides; that is, the pI of one of the monomers
(referred to herein for simplicity as "monomer A") can be
engineered away from monomer B, or both monomer A and B change be
changed, with the pI of monomer A increasing and the pI of monomer
B decreasing. As is outlined more fully below, the pI changes of
either or both monomers can be done by removing or adding a charged
residue (e.g. a neutral amino acid is replaced by a positively or
negatively charged amino acid residue, e.g. glycine to glutamic
acid), changing a charged residue from positive or negative to the
opposite charge (aspartic acid to lysine) or changing a charged
residue to a neutral residue (e.g. loss of a charge; lysine to
serine.). A number of these variants are shown in the Figures.
[0181] Accordingly, this embodiment of the present invention
provides for creating a sufficient change in pI in at least one of
the monomers such that heterodimers can be separated from
homodimers. As will be appreciated by those in the art, and as
discussed further below, this can be done by using a "wild type"
heavy chain constant region and a variant region that has been
engineered to either increase or decrease it's pI (wt A-+B or wt
A--B), or by increasing one region and decreasing the other region
(A+-B- or A-B+).
[0182] Thus, in general, a component of some embodiments of the
present invention are amino acid variants in the constant regions
of antibodies that are directed to altering the isoelectric point
(pI) of at least one, if not both, of the monomers of a dimeric
protein to form "pI antibodies") by incorporating amino acid
substitutions ("pI variants" or "pI substitutions") into one or
both of the monomers. As shown herein, the separation of the
heterodimers from the two homodimers can be accomplished if the pIs
of the two monomers differ by as little as 0.1 pH unit, with 0.2,
0.3, 0.4 and 0.5 or greater all finding use in the present
invention.
[0183] As will be appreciated by those in the art, the number of pI
variants to be included on each or both monomer(s) to get good
separation will depend in part on the starting pI of the
components, for example in the triple F format, the starting pI of
the scFv and Fab of interest. That is, to determine which monomer
to engineer or in which "direction" (e.g. more positive or more
negative), the Fv sequences of the two target antigens are
calculated and a decision is made from there. As is known in the
art, different Fvs will have different starting pIs which are
exploited in the present invention. In general, as outlined herein,
the pIs are engineered to result in a total pI difference of each
monomer of at least about 0.1 logs, with 0.2 to 0.5 being preferred
as outlined herein.
[0184] Furthermore, as will be appreciated by those in the art and
outlined herein, in some embodiments, heterodimers can be separated
from homodimers on the basis of size. As shown in FIG. 1 for
example, several of the formats allow separation of heterodimers
and homodimers on the basis of size.
[0185] In the case where pI variants are used to achieve
heterodimerization, by using the constant region(s) of the heavy
chain(s), a more modular approach to designing and purifying
bispecific proteins, including antibodies, is provided. Thus, in
some embodiments, heterodimerization variants (including skew and
purification heterodimerization variants) are not included in the
variable regions, such that each individual antibody must be
engineered. In addition, in some embodiments, the possibility of
immunogenicity resulting from the pI variants is significantly
reduced by importing pI variants from different IgG isotypes such
that pI is changed without introducing significant immunogenicity.
Thus, an additional problem to be solved is the elucidation of low
pI constant domains with high human sequence content, e.g. the
minimization or avoidance of non-human residues at any particular
position.
[0186] A side benefit that can occur with this pI engineering is
also the extension of serum half-life and increased FcRn binding.
That is, as described in U.S. Ser. No. 13/194,904 (incorporated by
reference in its entirety), lowering the pI of antibody constant
domains (including those found in antibodies and Fc fusions) can
lead to longer serum retention in vivo. These pI variants for
increased serum half life also facilitate pI changes for
purification.
[0187] In addition, it should be noted that the pI variants of the
heterodimerization variants give an additional benefit for the
analytics and quality control process of bispecific antibodies, as
the ability to either eliminate, minimize and distinguish when
homodimers are present is significant. Similarly, the ability to
reliably test the reproducibility of the heterodimeric antibody
production is important.
Heterodimerization Variants
[0188] The present invention provides heterodimeric proteins,
including heterodimeric antibodies in a variety of formats, which
utilize heterodimeric variants to allow for heterodimeric formation
and/or purification away from homodimers.
[0189] There are a number of suitable pairs of sets of
heterodimerization skew variants. These variants come in "pairs" of
"sets". That is, one set of the pair is incorporated into the first
monomer and the other set of the pair is incorporated into the
second monomer. It should be noted that these sets do not
necessarily behave as "knobs in holes" variants, with a one-to-one
correspondence between a residue on one monomer and a residue on
the other; that is, these pairs of sets form an interface between
the two monomers that encourages heterodimer formation and
discourages homodimer formation, allowing the percentage of
heterodimers that spontaneously form under biological conditions to
be over 90%, rather than the expected 50% (25% homodimer A/A:50%
heterodimer A/B:25% homodimer B/B).
Steric Variants
[0190] In some embodiments, the formation of heterodimers can be
facilitated by the addition of steric variants. That is, by
changing amino acids in each heavy chain, different heavy chains
are more likely to associate to form the heterodimeric structure
than to form homodimers with the same Fc amino acid sequences.
Suitable steric variants are included in FIG. 29.
[0191] One mechanism is generally referred to in the art as "knobs
and holes", referring to amino acid engineering that creates steric
influences to favor heterodimeric formation and disfavor
homodimeric formation can also optionally be used; this is
sometimes referred to as "knobs and holes", as described in U.S.
Ser. No. 61/596,846, Ridgway et al., Protein Engineering 9(7):617
(1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S. Pat. No.
8,216,805, all of which are hereby incorporated by reference in
their entirety. The Figures identify a number of "monomer A-monomer
B" pairs that rely on "knobs and holes". In addition, as described
in Merchant et al., Nature Biotech. 16:677 (1998), these "knobs and
hole" mutations can be combined with disulfide bonds to skew
formation to heterodimerization.
[0192] An additional mechanism that finds use in the generation of
heterodimers is sometimes referred to as "electrostatic steering"
as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637
(2010), hereby incorporated by reference in its entirety. This is
sometimes referred to herein as "charge pairs". In this embodiment,
electrostatics are used to skew the formation towards
heterodimerization. As those in the art will appreciate, these may
also have an effect on pI, and thus on purification, and thus could
in some cases also be considered pI variants. However, as these
were generated to force heterodimerization and were not used as
purification tools, they are classified as "steric variants". These
include, but are not limited to, D221E/P228E/L368E paired with
D221R/P228R/K409R (e.g. these are "monomer corresponding sets) and
C220E/P228E/368E paired with C220R/E224R/P228R/K409R.
[0193] Additional monomer A and monomer B variants that can be
combined with other variants, optionally and independently in any
amount, such as pI variants outlined herein or other steric
variants that are shown in FIG. 37 of US 2012/0149876, the figure
and legend and SEQ ID NOs of which are incorporated expressly by
reference herein.
[0194] In some embodiments, the steric variants outlined herein can
be optionally and independently incorporated with any pI variant
(or other variants such as Fc variants, FcRn variants, etc.) into
one or both monomers, and can be independently and optionally
included or excluded from the proteins of the invention.
[0195] A list of suitable skew variants is found in FIG. 29, with
FIG. 34 showing some pairs of particular utility in many
embodiments. Of particular use in many embodiments are the pairs of
sets including, but not limited to, S364K/E357Q:L368D/K370S;
L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;
L368D/K370S:S364K/E357L and K370S:S364K/E357Q. In terms of
nomenclature, the pair "S364K/E357Q:L368D/K370S" means that one of
the monomers has the double variant set S364K/E357Q and the other
has the double variant set L368D/K370S.
pI (Isoelectric Point) Variants for Heterodimers
[0196] In general, as will be appreciated by those in the art,
there are two general categories of pI variants: those that
increase the pI of the protein (basic changes) and those that
decrease the pI of the protein (acidic changes). As described
herein, all combinations of these variants can be done: one monomer
may be wild type, or a variant that does not display a
significantly different pI from wild-type, and the other can be
either more basic or more acidic. Alternatively, each monomer is
changed, one to more basic and one to more acidic.
[0197] Preferred combinations of pI variants are shown in FIG. 30.
As outlined herein and shown in the figures, these changes are
shown relative to IgG1, but all isotypes can be altered this way,
as well as isotype hybrids. In the case where the heavy chain
constant domain is from IgG2-4, R133E and R133Q can also be
used.
Antibody Heterodimers Light Chain Variants
[0198] In the case of antibody based heterodimers, e.g. where at
least one of the monomers comprises a light chain in addition to
the heavy chain domain, pI variants can also be made in the light
chain. Amino acid substitutions for lowering the pI of the light
chain include, but are not limited to, K126E, K126Q, K145E, K145Q,
N152D, S156E, K169E, S202E, K207E and adding peptide DEDE at the
c-terminus of the light chain. Changes in this category based on
the constant lambda light chain include one or more substitutions
at R108Q Q124E, K126Q, N138D, K145T and Q199E. In addition,
increasing the pI of the light chains can also be done.
Isotypic Variants
[0199] In addition, many embodiments of the invention rely on the
"importation" of pI amino acids at particular positions from one
IgG isotype into another, thus reducing or eliminating the
possibility of unwanted immunogenicity being introduced into the
variants. A number of these are shown in FIG. 21 of US Publ.
2014/0370013, hereby incorporated by reference. That is, IgG1 is a
common isotype for therapeutic antibodies for a variety of reasons,
including high effector function. However, the heavy constant
region of IgG1 has a higher pI than that of IgG2 (8.10 versus
7.31). By introducing IgG2 residues at particular positions into
the IgG1 backbone, the pI of the resulting monomer is lowered (or
increased) and additionally exhibits longer serum half-life. For
example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 has
a glutamic acid (pI 3.22); importing the glutamic acid will affect
the pI of the resulting protein. As is described below, a number of
amino acid substitutions are generally required to significant
affect the pI of the variant antibody. However, it should be noted
as discussed below that even changes in IgG2 molecules allow for
increased serum half-life.
[0200] In other embodiments, non-isotypic amino acid changes are
made, either to reduce the overall charge state of the resulting
protein (e.g. by changing a higher pI amino acid to a lower pI
amino acid), or to allow accommodations in structure for stability,
etc. as is more further described below.
[0201] In addition, by pI engineering both the heavy and light
constant domains, significant changes in each monomer of the
heterodimer can be seen. As discussed herein, having the pIs of the
two monomers differ by at least 0.5 can allow separation by ion
exchange chromatography or isoelectric focusing, or other methods
sensitive to isoelectric point.
Calculating pI
[0202] The pI of each monomer can depend on the pI of the variant
heavy chain constant domain and the pI of the total monomer,
including the variant heavy chain constant domain and the fusion
partner. Thus, in some embodiments, the change in pI is calculated
on the basis of the variant heavy chain constant domain, using the
chart in the FIG. 19 of US Pub. 2014/0370013. As discussed herein,
which monomer to engineer is generally decided by the inherent pI
of the Fv and scaffold regions. Alternatively, the pI of each
monomer can be compared.
pI Variants that Also Confer Better FcRn In Vivo Binding
[0203] In the case where the pI variant decreases the pI of the
monomer, they can have the added benefit of improving serum
retention in vivo.
[0204] Although still under examination, Fc regions are believed to
have longer half-lives in vivo, because binding to FcRn at pH 6 in
an endosome sequesters the Fc (Ghetie and Ward, 1997 Immunol Today.
18(12): 592-598, entirely incorporated by reference). The endosomal
compartment then recycles the Fc to the cell surface. Once the
compartment opens to the extracellular space, the higher pH,
.about.7.4, induces the release of Fc back into the blood. In mice,
Dall'Acqua et al. showed that Fc mutants with increased FcRn
binding at pH 6 and pH 7.4 actually had reduced serum
concentrations and the same half life as wild-type Fc (Dall'Acqua
et al. 2002, J. Immunol. 169:5171-5180, entirely incorporated by
reference). The increased affinity of Fc for FcRn at pH 7.4 is
thought to forbid the release of the Fc back into the blood.
Therefore, the Fc mutations that will increase Fc's half-life in
vivo will ideally increase FcRn binding at the lower pH while still
allowing release of Fc at higher pH. The amino acid histidine
changes its charge state in the pH range of 6.0 to 7.4. Therefore,
it is not surprising to find His residues at important positions in
the Fc/FcRn complex.
[0205] Recently it has been suggested that antibodies with variable
regions that have lower isoelectric points may also have longer
serum half-lives (Igawa et al., 2010 PEDS. 23(5): 385-392, entirely
incorporated by reference). However, the mechanism of this is still
poorly understood. Moreover, variable regions differ from antibody
to antibody. Constant region variants with reduced pI and extended
half-life would provide a more modular approach to improving the
pharmacokinetic properties of antibodies, as described herein.
Additional Fc Variants for Additional Functionality
[0206] In addition to pI amino acid variants, there are a number of
useful Fc amino acid modification that can be made for a variety of
reasons, including, but not limited to, altering binding to one or
more Fc.gamma.R receptors, altered binding to FcRn receptors,
etc.
[0207] Accordingly, the proteins of the invention can include amino
acid modifications, including the heterodimerization variants
outlined herein, which includes the pI variants and steric
variants. Each set of variants can be independently and optionally
included or excluded from any particular heterodimeric protein.
Fc.gamma.R Variants
[0208] Accordingly, there are a number of useful Fc substitutions
that can be made to alter binding to one or more of the Fc.gamma.R
receptors. Substitutions that result in increased binding as well
as decreased binding can be useful. For example, it is known that
increased binding to FcRIIIa generally results in increased ADCC
(antibody dependent cell-mediated cytotoxicity; the cell-mediated
reaction wherein nonspecific cytotoxic cells that express
Fc.gamma.Rs recognize bound antibody on a target cell and
subsequently cause lysis of the target cell). Similarly, decreased
binding to Fc.gamma.RIIb (an inhibitory receptor) can be beneficial
as well in some circumstances. Amino acid substitutions that find
use in the present invention include those listed in U.S. Ser. No.
11/124,620 (particularly FIG. 41), Ser. Nos. 11/174,287,
11/396,495, 11/538,406, all of which are expressly incorporated
herein by reference in their entirety and specifically for the
variants disclosed therein. Particular variants that find use
include, but are not limited to, 236A, 239D, 239E, 332E, 332D,
239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y,
239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.
[0209] In addition, there are additional Fc substitutions that find
use in increased binding to the FcRn receptor and increased serum
half life, as specifically disclosed in U.S. Ser. No. 12/341,769,
hereby incorporated by reference in its entirety, including, but
not limited to, 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F,
436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L.
Ablation Variants
[0210] Similarly, another category of functional variants are
"Fc.gamma.R ablation variants" or "Fc knock out (FcKO or KO)"
variants. In these embodiments, for some therapeutic applications,
it is desirable to reduce or remove the normal binding of the Fc
domain to one or more or all of the Fc.gamma. receptors (e.g.
Fc.gamma.R1, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIIa, etc.) to
avoid additional mechanisms of action. That is, for example, in
many embodiments, particularly in the use of bispecific antibodies
that bind CD3 monovalently it is generally desirable to ablate
Fc.gamma.RIIIa binding to eliminate or significantly reduce ADCC
activity. wherein one of the Fc domains comprises one or more
Fc.gamma. receptor ablation variants. These ablation variants are
depicted in FIG. 31, and each can be independently and optionally
included or excluded, with preferred aspects utilizing ablation
variants selected from the group consisting of G236R/L328R,
E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,
E233P/L234V/L235A/G236del/S239K/A327G,
E233P/L234V/L235A/G236del/S267K/A327G and
E233P/L234V/L235A/G236del. It should be noted that the ablation
variants referenced herein ablate Fc.gamma.R binding but generally
not FcRn binding.
Combination of Heterodimeric and Fc Variants
[0211] As will be appreciated by those in the art, all of the
recited heterodimerization variants (including skew and/or pI
variants) can be optionally and independently combined in any way,
as long as they retain their "strandedness" or "monomer partition".
In addition, all of these variants can be combined into any of the
heterodimerization formats.
[0212] In the case of pI variants, while embodiments finding
particular use are shown in the Figures, other combinations can be
generated, following the basic rule of altering the pI difference
between two monomers to facilitate purification.
[0213] In addition, any of the heterodimerization variants, skew
and pI, are also independently and optionally combined with Fc
ablation variants, Fc variants, FcRn variants, as generally
outlined herein.
Useful Formats of the Invention
[0214] As will be appreciated by those in the art and discussed
more fully below, the heterodimeric fusion proteins of the present
invention can take on a wide variety of configurations, as are
generally depicted in FIG. 1. Some figures depict "single ended"
configurations, where there is one type of specificity on one "arm"
of the molecule and a different specificity on the other "arm".
Other figures depict "dual ended" configurations, where there is at
least one type of specificity at the "top" of the molecule and one
or more different specificities at the "bottom" of the molecule.
Thus, the present invention is directed to novel immunoglobulin
compositions that co-engage a different first and a second
antigen.
[0215] As will be appreciated by those in the art, the
heterodimeric formats of the invention can have different valencies
as well as be bispecific. That is, heterodimeric antibodies of the
invention can be bivalent and bispecific, wherein CD3 is bound by
one binding domain and CD38 is bound by a second binding domain.
The heterodimeric antibodies can also be trivalent and bispecific,
wherein the CD38 is bound by two binding domains and the CD3 by a
second binding domain. As is outlined herein, it is preferable that
the CD3 is bound only monovalently, to reduce potential side
effects.
[0216] The present invention utilizes anti-CD3 antigen binding
domains and anti-CD38 antigen binding domains. As will be
appreciated by those in the art, any collection of anti-CD3 CDRs,
anti-CD3 variable light and variable heavy domains, Fabs and scFvs
as depicted in any of the Figures (see particularly FIGS. 2 through
7, and FIG. 68) can be used. Similarly, any of the anti-CD38
antigen binding domains, whether anti-CD38 CDRs, anti-CD38 variable
light and variable heavy domains, Fabs and scFvs as depicted in any
of the Figures (see FIGS. 8, 9 and 10) can be used, optionally and
independently combined in any combination.
Bottle Opener Format
[0217] One heterodimeric scaffold that finds particular use in the
present invention is the "triple F" or "bottle opener" scaffold
format as shown in FIGS. 1A, A and B. In this embodiment, one heavy
chain of the antibody contains an single chain Fv ("scFv", as
defined below) and the other heavy chain is a "regular" FAb format,
comprising a variable heavy chain and a light chain. This structure
is sometimes referred to herein as "triple F" format (scFv-FAb-Fc)
or the "bottle-opener" format, due to a rough visual similarity to
a bottle-opener (see FIG. 1). The two chains are brought together
by the use of amino acid variants in the constant regions (e.g. the
Fc domain, the CH1 domain and/or the hinge region) that promote the
formation of heterodimeric antibodies as is described more fully
below.
[0218] There are several distinct advantages to the present "triple
F" format. As is known in the art, antibody analogs relying on two
scFv constructs often have stability and aggregation problems,
which can be alleviated in the present invention by the addition of
a "regular" heavy and light chain pairing. In addition, as opposed
to formats that rely on two heavy chains and two light chains,
there is no issue with the incorrect pairing of heavy and light
chains (e.g. heavy 1 pairing with light 2, etc.).
[0219] Many of the embodiments outlined herein rely in general on
the bottle opener format that comprises a first monomer comprising
an scFv, comprising a variable heavy and a variable light domain,
covalently attached using an scFv linker (charged, in many
instances), where the scFv is covalently attached to the N-terminus
of a first Fc domain usually through a domain linker (which, as
outlined herein can either be un-charged or charged). The second
monomer of the bottle opener format is a heavy chain, and the
composition further comprises a light chain.
[0220] In general, in many preferred embodiments, the scFv is the
domain that binds to the CD3, with the Fab of the heavy and light
chains binding to CD38. In addition, the Fc domains of the
invention generally comprise skew variants (e.g. a set of amino
acid substitutions as shown in FIG. 29 and FIG. 34, with
particularly useful skew variants being selected from the group
consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;
L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L
and K370S:S364K/E357Q), optionally ablation variants, and the heavy
chain comprises pI variants.
[0221] The present invention provides bottle opener formats where
the anti-CD3 scFv sequences are as shown in FIGS. 2 to 7 and FIG.
68.
[0222] The present invention provides bottle opener formats wherein
the anti-CD38 sequences are as shown in FIGS. 8 to 10.
mAb-Fv Format
[0223] One heterodimeric scaffold that finds particular use in the
present invention is the mAb-Fv format shown in FIG. 1. In this
embodiment, the format relies on the use of a C-terminal attachment
of an "extra" variable heavy domain to one monomer and the
C-terminal attachment of an "extra" variable light domain to the
other monomer, thus forming a third antigen binding domain, wherein
the Fab portions of the two monomers bind CD38 and the "extra" scFv
domain binds CD3.
[0224] In this embodiment, the first monomer comprises a first
heavy chain, comprising a first variable heavy domain and a first
constant heavy domain comprising a first Fc domain, with a first
variable light domain covalently attached to the C-terminus of the
first Fc domain using a domain linker. The second monomer comprises
a second variable heavy domain of the second constant heavy domain
comprising a second Fc domain, and a third variable heavy domain
covalently attached to the C-terminus of the second Fc domain using
a domain linker. The two C-terminally attached variable domains
make up a scFv that binds CD3. This embodiment further utilizes a
common light chain comprising a variable light domain and a
constant light domain, that associates with the heavy chains to
form two identical Fabs that bind CD38. As for many of the
embodiments herein, these constructs include skew variants, pI
variants, ablation variants, additional Fc variants, etc. as
desired and described herein.
[0225] The present invention provides mAb-Fv formats where the
anti-CD3 scFv sequences are as shown in FIGS. 2 to 7.
[0226] The present invention provides mAb-Fv formats wherein the
anti-CD38 sequences are as shown in FIGS. 8 to 10.
[0227] The present invention provides mAb-Fv formats comprising
ablation variants as shown in FIG. 31.
[0228] The present invention provides mAb-Fv formats comprising
skew variants as shown in FIGS. 29 and 34.
mAb-scFv
[0229] One heterodimeric scaffold that finds particular use in the
present invention is the mAb-Fv format shown in FIG. 1. In this
embodiment, the format relies on the use of a C-terminal attachment
of a scFv to one of the monomers, thus forming a third antigen
binding domain, wherein the Fab portions of the two monomers bind
CD38 and the "extra" scFv domain binds CD3. Thus, the first monomer
comprises a first heavy chain (comprising a variable heavy domain
and a constant domain), with a C-terminally covalently attached
scFv comprising a scFv variable light domain, an scFv linker and a
scFv variable heavy domain. This embodiment further utilizes a
common light chain comprising a variable light domain and a
constant light domain, that associates with the heavy chains to
form two identical Fabs that bind CD38. As for many of the
embodiments herein, these constructs include skew variants, pI
variants, ablation variants, additional Fc variants, etc. as
desired and described herein.
[0230] The present invention provides mAb-scFv formats where the
anti-CD3 scFv sequences are as shown in FIGS. 2 to 7.
[0231] The present invention provides mAb-scFv formats wherein the
anti-CD38 sequences are as shown in FIGS. 8 to 10.
[0232] The present invention provides mAb-scFv formats comprising
ablation variants as shown in FIG. 31.
[0233] The present invention provides mAb-scFv formats comprising
skew variants as shown in FIGS. 29 and 34.
Central scFv
[0234] One heterodimeric scaffold that finds particular use in the
present invention is the Central-scFv format shown in FIG. 1. In
this embodiment, the format relies on the use of an inserted scFv
domain thus forming a third antigen binding domain, wherein the Fab
portions of the two monomers bind CD38 and the "extra" scFv domain
binds CD3. The scFv domain is inserted between the Fc domain and
the CH1-Fv region of one of the monomers, thus providing a third
antigen binding domain.
[0235] In this embodiment, one monomer comprises a first heavy
chain comprising a first variable heavy domain, a CH1 domain and Fc
domain, with a scFv comprising a scFv variable light domain, an
scFv linker and a scFv variable heavy domain. The scFv is
covalently attached between the C-terminus of the CH1 domain of the
heavy constant domain and the N-terminus of the first Fc domain
using domain linkers. This embodiment further utilizes a common
light chain comprising a variable light domain and a constant light
domain, that associates with the heavy chains to form two identical
Fabs that bind CD38. As for many of the embodiments herein, these
constructs include skew variants, pI variants, ablation variants,
additional Fc variants, etc. as desired and described herein.
[0236] The present invention provides Central-scFv formats where
the anti-CD3 scFv sequences are as shown in FIGS. 2 to 7.
[0237] The present invention provides Central-scFv formats wherein
the anti-CD38 sequences are as shown in FIGS. 8 to 10.
[0238] The present invention provides Central-scFv formats
comprising ablation variants as shown in FIG. 31.
[0239] The present invention provides Central-scFv formats
comprising skew variants as shown in FIGS. 29 and 34.
Central-Fv Format
[0240] One heterodimeric scaffold that finds particular use in the
present invention is the Central-Fv format shown in FIG. 1. In this
embodiment, the format relies on the use of an inserted scFv domain
thus forming a third antigen binding domain, wherein the Fab
portions of the two monomers bind CD38 and the "extra" scFv domain
binds CD3. The scFv domain is inserted between the Fc domain and
the CH1-Fv region of the monomers, thus providing a third antigen
binding domain, wherein each monomer contains a component of the
scFv (e.g. one monomer comprises a variable heavy domain and the
other a variable light domain).
[0241] In this embodiment, one monomer comprises a first heavy
chain comprising a first variable heavy domain, a CH1 domain and Fc
domain and an additional variable light domain. The light domain is
covalently attached between the C-terminus of the CH1 domain of the
heavy constant domain and the N-terminus of the first Fc domain
using domain linkers. The other monomer comprises a first heavy
chain comprising a first variable heavy domain, a CH1 domain and Fc
domain and an additional variable heavy domain. The light domain is
covalently attached between the C-terminus of the CH1 domain of the
heavy constant domain and the N-terminus of the first Fc domain
using domain linkers.
[0242] This embodiment further utilizes a common light chain
comprising a variable light domain and a constant light domain,
that associates with the heavy chains to form two identical Fabs
that bind CD38. As for many of the embodiments herein, these
constructs include skew variants, pI variants, ablation variants,
additional Fc variants, etc. as desired and described herein.
[0243] The present invention provides Central-scFv formats where
the anti-CD3 scFv sequences are as shown in FIGS. 2 to 7.
[0244] The present invention provides Central-scFv formats wherein
the anti-CD38 sequences are as shown in FIGS. 8 to 10.
[0245] The present invention provides Central-scFv formats
comprising ablation variants as shown in FIG. 31.
[0246] The present invention provides Central-scFv formats
comprising skew variants as shown in FIGS. 29 and 34.
One Armed Central-scFv
[0247] One heterodimeric scaffold that finds particular use in the
present invention is the one armed central-scFv format shown in
FIG. 1. In this embodiment, one monomer comprises just an Fc
domain, while the other monomer uses an inserted scFv domain thus
forming the second antigen binding domain. In this format, either
the Fab portion binds CD38 and the scFv binds CD3 or vice versa.
The scFv domain is inserted between the Fc domain and the CH1-Fv
region of one of the monomers.
[0248] In this embodiment, one monomer comprises a first heavy
chain comprising a first variable heavy domain, a CH1 domain and Fc
domain, with a scFv comprising a scFv variable light domain, an
scFv linker and a scFv variable heavy domain. The scFv is
covalently attached between the C-terminus of the CH1 domain of the
heavy constant domain and the N-terminus of the first Fc domain
using domain linkers. The second monomer comprises an Fc domain.
This embodiment further utilizes a light chain comprising a
variable light domain and a constant light domain, that associates
with the heavy chain to form a Fab. As for many of the embodiments
herein, these constructs include skew variants, pI variants,
ablation variants, additional Fc variants, etc. as desired and
described herein.
[0249] The present invention provides one armed Central-scFv
formats where the anti-CD3 scFv sequences are as shown in FIGS. 2
to 7.
[0250] The present invention provides one armed Central-scFv
formats wherein the anti-CD38 sequences are as shown in FIGS. 8 to
10.
[0251] The present invention provides one armed Central-scFv
formats comprising ablation variants as shown in FIG. 31.
[0252] The present invention provides one armed Central-scFv
formats comprising skew variants as shown in FIGS. 29 and 34.
Dual scFv Formats
[0253] The present invention also provides dual scFv formats as are
known in the art and shown in FIG. 1. In particular, the invention
provides dual scFv formats where the anti-CD3 scFv sequences are as
shown in FIGS. 2 to 7.
[0254] The present invention provides dual scFv formats wherein the
anti-CD38 sequences are as shown in FIGS. 8 to 10.
Nucleic Acids of the Invention
[0255] The invention further provides nucleic acid compositions
encoding the bispecific antibodies of the invention. As will be
appreciated by those in the art, the nucleic acid compositions will
depend on the format and scaffold of the heterodimeric protein.
Thus, for example, when the format requires three amino acid
sequences, such as for the triple F format (e.g. a first amino acid
monomer comprising an Fc domain and a scFv, a second amino acid
monomer comprising a heavy chain and a light chain), three nucleic
acid sequences can be incorporated into one or more expression
vectors for expression. Similarly, some formats (e.g. dual scFv
formats such as disclosed in FIG. 1) only two nucleic acids are
needed; again, they can be put into one or two expression
vectors.
[0256] As is known in the art, the nucleic acids encoding the
components of the invention can be incorporated into expression
vectors as is known in the art, and depending on the host cells
used to produce the heterodimeric antibodies of the invention.
Generally the nucleic acids are operably linked to any number of
regulatory elements (promoters, origin of replication, selectable
markers, ribosomal binding sites, inducers, etc.). The expression
vectors can be extra-chromosomal or integrating vectors.
[0257] The nucleic acids and/or expression vectors of the invention
are then transformed into any number of different types of host
cells as is well known in the art, including mammalian, bacterial,
yeast, insect and/or fungal cells, with mammalian cells (e.g. CHO
cells), finding use in many embodiments.
[0258] In some embodiments, nucleic acids encoding each monomer and
the optional nucleic acid encoding a light chain, as applicable
depending on the format, are each contained within a single
expression vector, generally under different or the same promoter
controls. In embodiments of particular use in the present
invention, each of these two or three nucleic acids are contained
on a different expression vector. As shown herein and in
62/025,931, hereby incorporated by reference, different vector
ratios can be used to drive heterodimer formation. That is,
surprisingly, while the proteins comprise first monomer:second
monomer:light chains (in the case of many of the embodiments herein
that have three polypeptides comprising the heterodimeric antibody)
in a 1:1:2 ratio, these are not the ratios that give the best
results. See FIG. 65.
[0259] The heterodimeric antibodies of the invention are made by
culturing host cells comprising the expression vector(s) as is well
known in the art. Once produced, traditional antibody purification
steps are done, including an ion exchange chromotography step. As
discussed herein, having the pIs of the two monomers differ by at
least 0.5 can allow separation by ion exchange chromatography or
isoelectric focusing, or other methods sensitive to isoelectric
point. That is, the inclusion of pI substitutions that alter the
isoelectric point (pI) of each monomer so that such that each
monomer has a different pI and the heterodimer also has a distinct
pI, thus facilitating isoelectric purification of the "triple F"
heterodimer (e.g., anionic exchange columns, cationic exchange
columns). These substitutions also aid in the determination and
monitoring of any contaminating dual scFv-Fc and mAb homodimers
post-purification (e.g., IEF gels, cIEF, and analytical IEX
columns).
Treatments
[0260] Once made, the compositions of the invention find use in a
number of applications. CD38 is unregulated in many hematopoeitic
malignancies and in cell lines derived from various hematopoietic
malignancies including non-Hodgkin's lymphoma (NHL), Burkitt's
lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic
leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell
lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia
(HCL), Hodgkin's Lymphoma (HL), chronic lymphocytic leukemia (CLL)
and chronic myeloid leukemia (CML).
[0261] Accordingly, the heterodimeric compositions of the invention
find use in the treatment of these cancers.
Antibody Compositions for In Vivo Administration
[0262] Formulations of the antibodies used in accordance with the
present invention are prepared for storage by mixing an antibody
having the desired degree of purity with optional pharmaceutically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the
form of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and include buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0263] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
provide antibodies with other specificities. Alternatively, or in
addition, the composition may comprise a cytotoxic agent, cytokine,
growth inhibitory agent and/or small molecule antagonist. Such
molecules are suitably present in combination in amounts that are
effective for the purpose intended.
[0264] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0265] The formulations to be used for in vivo administration
should be sterile, or nearly so. This is readily accomplished by
filtration through sterile filtration membranes.
[0266] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0267] When encapsulated antibodies remain in the body for a long
time, they may denature or aggregate as a result of exposure to
moisture at 37.degree. C., resulting in a loss of biological
activity and possible changes in immunogenicity. Rational
strategies can be devised for stabilization depending on the
mechanism involved. For example, if the aggregation mechanism is
discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
Administrative Modalities
[0268] The antibodies and chemotherapeutic agents of the invention
are administered to a subject, in accord with known methods, such
as intravenous administration as a bolus or by continuous infusion
over a period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. Intravenous or
subcutaneous administration of the antibody is preferred.
Treatment Modalities
[0269] In the methods of the invention, therapy is used to provide
a positive therapeutic response with respect to a disease or
condition. By "positive therapeutic response" is intended an
improvement in the disease or condition, and/or an improvement in
the symptoms associated with the disease or condition. For example,
a positive therapeutic response would refer to one or more of the
following improvements in the disease: (1) a reduction in the
number of neoplastic cells; (2) an increase in neoplastic cell
death; (3) inhibition of neoplastic cell survival; (5) inhibition
(i.e., slowing to some extent, preferably halting) of tumor growth;
(6) an increased patient survival rate; and (7) some relief from
one or more symptoms associated with the disease or condition.
[0270] Positive therapeutic responses in any given disease or
condition can be determined by standardized response criteria
specific to that disease or condition. Tumor response can be
assessed for changes in tumor morphology (i.e., overall tumor
burden, tumor size, and the like) using screening techniques such
as magnetic resonance imaging (MRI) scan, x-radiographic imaging,
computed tomographic (CT) scan, bone scan imaging, endoscopy, and
tumor biopsy sampling including bone marrow aspiration (BMA) and
counting of tumor cells in the circulation.
[0271] In addition to these positive therapeutic responses, the
subject undergoing therapy may experience the beneficial effect of
an improvement in the symptoms associated with the disease.
[0272] An improvement in the disease may be characterized as a
complete response. By "complete response" is intended an absence of
clinically detectable disease with normalization of any previously
abnormal radiographic studies, bone marrow, and cerebrospinal fluid
(CSF) or abnormal monoclonal protein in the case of myeloma.
[0273] Such a response may persist for at least 4 to 8 weeks, or
sometimes 6 to 8 weeks, following treatment according to the
methods of the invention. Alternatively, an improvement in the
disease may be categorized as being a partial response. By "partial
response" is intended at least about a 50% decrease in all
measurable tumor burden (i.e., the number of malignant cells
present in the subject, or the measured bulk of tumor masses or the
quantity of abnormal monoclonal protein) in the absence of new
lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.
[0274] Treatment according to the present invention includes a
"therapeutically effective amount" of the medicaments used. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve a desired
therapeutic result.
[0275] A therapeutically effective amount may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the medicaments to elicit a desired
response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody
or antibody portion are outweighed by the therapeutically
beneficial effects.
[0276] A "therapeutically effective amount" for tumor therapy may
also be measured by its ability to stabilize the progression of
disease. The ability of a compound to inhibit cancer may be
evaluated in an animal model system predictive of efficacy in human
tumors.
[0277] Alternatively, this property of a composition may be
evaluated by examining the ability of the compound to inhibit cell
growth or to induce apoptosis by in vitro assays known to the
skilled practitioner. A therapeutically effective amount of a
therapeutic compound may decrease tumor size, or otherwise
ameliorate symptoms in a subject. One of ordinary skill in the art
would be able to determine such amounts based on such factors as
the subject's size, the severity of the subject's symptoms, and the
particular composition or route of administration selected.
[0278] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. Parenteral compositions may be formulated in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited
as unitary dosages for the subjects to be treated; each unit
contains a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0279] The specification for the dosage unit forms of the present
invention are dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0280] The efficient dosages and the dosage regimens for the
bispecific antibodies used in the present invention depend on the
disease or condition to be treated and may be determined by the
persons skilled in the art.
[0281] An exemplary, non-limiting range for a therapeutically
effective amount of an bispecific antibody used in the present
invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for
example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for
instance about 0.5, about such as 0.3, about 1, or about 3 mg/kg.
In another embodiment, he antibody is administered in a dose of 1
mg/kg or more, such as a dose of from 1 to 20 mg/kg, e.g. a dose of
from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.
[0282] A medical professional having ordinary skill in the art may
readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, a physician or a
veterinarian could start doses of the medicament employed in the
pharmaceutical composition at levels lower than that required in
order to achieve the desired therapeutic effect and gradually
increase the dosage until the desired effect is achieved.
[0283] In one embodiment, the bispecific antibody is administered
by infusion in a weekly dosage of from 10 to 500 mg/kg such as of
from 200 to 400 mg/kg Such administration may be repeated, e.g., 1
to 8 times, such as 3 to 5 times. The administration may be
performed by continuous infusion over a period of from 2 to 24
hours, such as of from 2 to 12 hours.
[0284] In one embodiment, the bispecific antibody is administered
by slow continuous infusion over a long period, such as more than
24 hours, if required to reduce side effects including
toxicity.
[0285] In one embodiment the bispecific antibody is administered in
a weekly dosage of from 250 mg to 2000 mg, such as for example 300
mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times,
such as from 4 to 6 times. The administration may be performed by
continuous infusion over a period of from 2 to 24 hours, such as of
from 2 to 12 hours. Such regimen may be repeated one or more times
as necessary, for example, after 6 months or 12 months. The dosage
may be determined or adjusted by measuring the amount of compound
of the present invention in the blood upon administration by for
instance taking out a biological sample and using anti-idiotypic
antibodies which target the antigen binding region of the
bispecific antibody.
[0286] In a further embodiment, the bispecific antibody is
administered once weekly for 2 to 12 weeks, such as for 3 to 10
weeks, such as for 4 to 8 weeks.
[0287] In one embodiment, the bispecific antibody is administered
by maintenance therapy, such as, e.g., once a week for a period of
6 months or more.
[0288] In one embodiment, the bispecific antibody is administered
by a regimen including one infusion of an bispecific antibody
followed by an infusion of an bispecific antibody conjugated to a
radioisotope. The regimen may be repeated, e.g., 7 to 9 days
later.
[0289] As non-limiting examples, treatment according to the present
invention may be provided as a daily dosage of an antibody in an
amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90
or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40,
or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of
treatment, or any combination thereof, using single or divided
doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination
thereof.
[0290] In some embodiments the bispecific antibody molecule thereof
is used in combination with one or more additional therapeutic
agents, e.g. a chemotherapeutic agent. Non-limiting examples of DNA
damaging chemotherapeutic agents include topoisomerase I inhibitors
(e.g., irinotecan, topotecan, camptothecin and analogs or
metabolites thereof, and doxorubicin); topoisomerase II inhibitors
(e.g., etoposide, teniposide, and daunorubicin); alkylating agents
(e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide,
carmustine, lomustine, semustine, streptozocin, decarbazine,
methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators
(e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators
and free radical generators such as bleomycin; and nucleoside
mimetics (e.g., 5-fluorouracil, capecitibine, gemcitabine,
fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin,
and hydroxyurea).
[0291] Chemotherapeutic agents that disrupt cell replication
include: paclitaxel, docetaxel, and related analogs; vincristine,
vinblastin, and related analogs; thalidomide, lenalidomide, and
related analogs (e.g., CC-5013 and CC-4047); protein tyrosine
kinase inhibitors (e.g., imatinib mesylate and gefitinib);
proteasome inhibitors (e.g., bortezomib); NF-.kappa.B inhibitors,
including inhibitors of I.kappa.B kinase; antibodies which bind to
proteins overexpressed in cancers and thereby downregulate cell
replication (e.g., trastuzumab, rituximab, cetuximab, and
bevacizumab); and other inhibitors of proteins or enzymes known to
be upregulated, over-expressed or activated in cancers, the
inhibition of which downregulates cell replication.
[0292] In some embodiments, the antibodies of the invention can be
used prior to, concurrent with, or after treatment with
Velcade.RTM. (bortezomib).
[0293] All cited references are herein expressly incorporated by
reference in their entirety.
[0294] Whereas particular embodiments of the invention have been
described above for purposes of illustration, it will be
appreciated by those skilled in the art that numerous variations of
the details may be made without departing from the invention as
described in the appended claims.
EXAMPLES
[0295] Examples are provided below to illustrate the present
invention. These examples are not meant to constrain the present
invention to any particular application or theory of operation. For
all constant region positions discussed in the present invention,
numbering is according to the EU index as in Kabat (Kabat et al.,
1991, Sequences of Proteins of Immunological Interest, 5th Ed.,
United States Public Health Service, National Institutes of Health,
Bethesda, entirely incorporated by reference). Those skilled in the
art of antibodies will appreciate that this convention consists of
nonsequential numbering in specific regions of an immunoglobulin
sequence, enabling a normalized reference to conserved positions in
immunoglobulin families. Accordingly, the positions of any given
immunoglobulin as defined by the EU index will not necessarily
correspond to its sequential sequence.
[0296] General and specific scientific techniques are outlined in
US Publications 2015/0307629, 2014/0288275 and WO2014/145806, all
of which are expressly incorporated by reference in their entirety
and particularly for the techniques outlined therein.
EXAMPLES
Example 1
Alternate Formats
[0297] Fab-scFv-Fc Production
[0298] DNA encoding the three chains needed for Fab-scFv-Fc
expression--Fab-Fc, scFv-Fc, and LC--were generated by gene
synthesis (Blue Heron Biotechnology, Bothell, Wash.) and were
subcloned using standard molecular biology techniques into the
expression vector pTT5. Substitutions were introduced using either
site-directed mutagenesis (QuikChange, Stratagene, Cedar Creek,
Tex.) or additional gene synthesis and subcloning. DNA was
transfected into HEK293E cells for expression and resulting
proteins were purified from the supernatant using protein A
affinity (GE Healthcare) and cation exchange (GE Healthcare)
chromatography. Amino acid sequences for Fab-scFv-Fc bispecifics
are listed in FIG. 3.
[0299] Surface Plasmon Resonance Affinity Determination
[0300] Surface plasmon resonance binding experiments were performed
using a Biacore 3000 instrument (data not shown). Even after amino
acids substitution(s) to modulate affinity, the anti-CD3 variable
region remains cross-reactive for cynomolgus monkey CD3.
[0301] Cell Surface Binding
[0302] Binding of Fab-scFv-Fcs to CD3 was measured on T cells via
detection with a secondary antibody.
[0303] Redirected T Cell Cytotoxicity
[0304] Anti-CD38.times.anti-CD3 Fab-scFv-Fc bispecifics were
characterized in vitro for redirected T cell cytotoxicity (RTCC) of
the CD20.sup.+ Ramos Burkitt's lymphoma (BL) cell line, CD20.sup.+
Jeko-1 Mantle Cell Lymphoma (MCL) cell line, and the CD38.sup.+
RPMI 8266 myeloma cell line. RTCC was measured and IL-6 production
during RTCC was also characterized (data not shown).
[0305] huPBL-SCID Immunoglobulin-Depletion Mouse Studies
[0306] The ability of anti-CD38.times.anti-CD3 Fab-scFv-Fc
bispecifics to deplete human immunoglobulins via depletion of human
B cells or plasma cells was assessed using human PBMC engrafted
SCID mice. Results are shown in the Figures.
Example 2
Alternate Formats
Bispecifics Production
[0307] Cartoon schematics of anti-CD38.times.anti-CD3 bispecifics
are shown in FIG. 1. Amino acid sequences for alternate format
anti-CD38.times.anti-CD3 bispecifics are listed in FIG. 39 to FIG.
43. DNA encoding the three chains needed for bispecific expression
were generated by gene synthesis (Blue Heron Biotechnology,
Bothell, Wash.) and were subcloned using standard molecular biology
techniques into the expression vector pTT5. Substitutions were
introduced using either site-directed mutagenesis (QuikChange,
Stratagene, Cedar Creek, Tex.) or additional gene synthesis and
subcloning. DNA was transfected into HEK293E cells for expression
and resulting proteins were purified from the supernatant using
protein A affinity (GE Healthcare) and cation exchange
chromatography. Yields following protein A affinity purification
are shown in FIG. 35. Cation exchange chromatography purification
was performed using a HiTrap SP HP column (GE Healthcare) with a
wash/equilibration buffer of 50 mM MES, pH 6.0 and an elution
buffer of 50 mM MES, pH 6.0+1 M NaCl linear gradient (see FIG. 36
for chromatograms).
Redirected T Cell Cytotoxicity
[0308] Anti-CD38.times.anti-CD3 bispecifics were characterized in
vitro for redirected T cell cytotoxicity (RTCC) of the CD38.sup.+
RPMI8266 myeloma cell line. 10 k RPMI8266 cells were incubated for
24 h with 500 k human PBMCs. RTCC was measured by LDH fluorescence
as indicated (see FIG. 37).
Example 3
Redirected T Cell Cytotoxicity
[0309] Anti-CD38.times.anti-CD3 Fab-scFv-Fc bispecifics were
characterized in vitro for redirected T cell cytotoxicity (RTCC) of
the CD38+ RPMI8266 myeloma cell line. 40 k RPMI8266 cells were
incubated for 96 h with 400 k human PBMCs. RTCC was measured by
flow cytometry as indicated (see FIG. 44). CD4+ and CD8+ T cell
expression of CD69, Ki-67, and PI-9 were also characterized by flow
cytometry and are shown in FIG. 45.
Mouse Model of Anti-Tumor Activity
[0310] Four groups of five NOD scid gamma (NSG) mice each were
engrafted with 5.times.106 RPMI8226TrS tumor cells (multiple
myeloma, luciferase-expressing) by intravenous tail vein injection
on Day -23. On Day 0, mice were engrafted intraperitoneally with
10.times.106 human PBMCs. After PBMC engraftment on Day 0, test
articles are dosed weekly (Days 0, 7) by intraperitoneal injection
at dose levels indicated in FIG. 4. Study design is further
summarized in FIG. 46. Tumor growth was monitored by measuring
total flux per mouse using an in vivo imaging system (IVIS.RTM.).
Both XmAb13551 and XmAb15426 showed substantial anti-tumor effects
(see FIG. 47 and FIG. 48).
Studies in Cynomolgus Monkey
[0311] Cynomolgus monkeys were given a single dose of
anti-CD38.times.anti-CD3 bispecifics. An anti-RSV.times.anti-CD3
bispecific control was also included. Dose levels were: 20 .mu.g/kg
XmAb13551 (n=2), 0.5 mg/kg XmAb15426 (n=3), 3 mg/kg XmAb14702
(n=3), or 3 mg/kg XmAb13245 (anti-RSV.times.anti-CD3 control, n=3)
(in 3 independent studies). Anti-CD38.times.anti-CD3 bispecifics
rapidly depleted CD38+ cells in peripheral blood (see FIG. 49).
Anti-CD38.times.anti-CD3 bispecifics resulted in T cell activation
as measured by CD69 expression (see FIG. 50). Serum levels of IL-6
were also measured (see FIG. 51). Note that, compared to XmAb13551,
XmAb15426 had an increased duration of CD38+ cell depletion and
lower levels of T cell activation and IL-6 production.
[0312] XmAb15426 and XmAb14702 were tested at single doses of 0.5
mg/kg and 3 mg/kg respectively. Both antibodies were well-tolerated
at these higher doses, consistent with the moderate levels of IL6
observed in serum from the treated monkeys. Moreover, XmAb15426,
with intermediate CD3 affinity, more effectively depleted CD38+
cells at 0.5 mg/kg compared to the original high-affinity XmAb13551
dosed at 2, 5 or 20 .mu.g/kg. Depletion by XmAb15426 was more
sustained compared to the highest dose of XmAb13551 in the previous
study (7 vs. 2 days, respectively). Notably, although target cell
depletion was greater for XmAb15426, T cell activation (CD69, CD25
and PD1 induction) was much lower in monkeys treated with XmAb15426
even dosed 25-fold higher than the 20 .mu.g/kg XmAb13551 group.
XmAb14702, with very low CD3 affinity, had little effect on CD38+
cells and T cell activation.
[0313] These results demonstrate that modulating T cell activation
by attenuating CD3 affinity is a promising method to improve the
therapeutic window of T cell-engaging bispecific antibodies. This
strategy has potential to expand the set of antigens amenable to
targeted T cell immunotherapy by improving tolerability and
enabling higher dosing to overcome antigen sink clearance with
targets such as CD38. We have shown that by reducing affinity for
CD3, XmAb 15426 effectively depletes CD38+ cells while minimizing
the CRS effects when with comparable doses of its high-affinity
counterpart XmAb13551.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160215063A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160215063A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References